Identification of Modulators of Autophagy

ABSTRACT

Methods for identifying compounds that inhibit or stimulate the autophagy pathway are described. Devices for detecting the expression of autophagy-related genes and kits for assaying the expression of autophagy-related genes are also described. Also described are methods for identifying individuals susceptible to or afflicted with a disease state associated with an autophagy pathway defect.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a Continuation of U.S. patent application Ser. No. 13/422,033, filed Mar. 16, 2012, which is a Continuation of U.S. patent application Ser. No. 13/284,923, filed Oct. 30, 2011, which is a Continuation of U.S. patent application Ser. No. 13/046,033, filed Mar. 11, 2011, which claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application Ser. No. 61/313,097, filed Mar. 11, 2010. U.S. patent application Ser. No. 13/046,033 is also a Continuation-in-Part of U.S. patent application Ser. No. 12/622,410, filed Nov. 19, 2009, which claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application Ser. No. 61/116,085, filed Nov. 19, 2008. The disclosures of each of the foregoing applications are hereby incorporated herein by reference in their entireties.

GOVERNMENT SUPPORT

The present application was supported in part by the National Institutes of Health under Grant Nos. R37 CA53370 and RO1 CA130893 and the Department of Defense under DOD W81XWH06-1-0514 and DOD W81XWH05. The U.S. government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Autophagy is a catabolic, cellular self-digestion process that is activated by starvation and stress whereby double membrane vesicles called autophagosomes form that engulf proteins and organelles. Autophagosomes then fuse with lysosomes where their cargo is degraded. The function of autophagy is to recycle intracellular nutrients to sustain metabolism during nutrient and growth factor deprivation, and to clear damaged proteins and organelles that accumulate during stress. Although elimination of individual proteins occurs by the ubiquitin-mediated proteasome degradation pathway, only the autophagy pathway can eliminate protein aggregates and organelles. Thus, autophagy complements and overlaps with proteasome function to prevent the accumulation of damaged cellular components during starvation and stress. Through these functions, autophagy is an important cellular stress response that functions to maintain protein and organelle quality control, protect the genome from damage, and sustain cell and mammalian viability.

Autophagy is controlled by ATG proteins that were initially identified in yeast for which there are mammalian homologues. ATG proteins are comprised of kinases, proteases, and two ubiquitin-like conjugation systems that likely function in concert with a host of unknown cellular proteins to control autophagosome formation, cargo recognition, engulfment, and trafficking to lysosomes. The ATG6/Beclin1-VPS34-ATG8/LC3 complex regulates autophagosome formation and LC3 cleavage, lipidation, and membrane translocation are frequently utilized to monitor autophagy induction and inhibition of flux through the autophagy pathway.

Targeting of cargo, including proteins and organelles, to autophagosomes for degradation is accomplished by tagging proteins with polyubiquitin. The ubiquitin-binding domain (UBA) on the adaptor protein p62 recognizes and binds these polyubiquitinated proteins. p62 oligomerizes by self-association of its PB1 domain and binds ATG8/LC3 on autophagosome membranes. p62 thereby identifies, collects and delivers cargo to autophagosomes for degradation. p62 itself is an autophagy substrate and is degraded by autophagy along with the cargo. As such, p62 accumulation in aggregates is indicative of autophagy inhibition and clearance of p62 following stress is indicative of functional autophagy. These properties of p62 have been demonstrated in vivo in autophagy-defective mutant mice and are mimicked by expression of EGFP-p62 in cell lines in vitro and in vivo (Mathew, R et al., (2009) Cell 137, 1062-1075).

The activation of autophagy by starvation and stress is controlled in part through the PI-3 kinase pathway via the protein kinase mTOR. Growth factor and nutrient availability promote mTOR activation that suppresses autophagy, whereas starvation and mTOR inactivation stimulate autophagy. While there are other mechanisms to regulate autophagy, and those that activate autophagy in response to stress are particularly poorly understood, mTOR provides a link between nutrient and growth factor availability, growth control, autophagy, and metabolism.

Autophagy dysfunction is believed to be a major contributor to human diseases including neurodegeneration, liver disease, and cancer. Many human neurodegenerative diseases are associated with aberrant protein accumulation and excessive neuronal cell death, and neurons of mice with targeted autophagy defects accumulate polyubiquitinated- and p62-containing protein aggregates that result in neurodegeneration. The human liver disease steatohepatitis and a major subset of hepatocellular carcinomas (HCCs) are associated with the formation of p62-containing protein aggregates (Mallory bodies), and livers of mice with autophagy defects have p62-containing protein aggregates, excessive cell death, and HCC.

Evidence from model organism disease models indicates that promoting autophagy with mTOR inhibitors such as rapamycin or CCI-779, and enhancing the clearance of misfolded, damaged or mutated proteins and protein aggregates prevents neurodegeneration, but that there also are mTOR-independent means to increase autophagy. Similarly, genetically eliminating the expression of p62 in hepatocytes and preventing p62 accumulation in autophagy-defective atg7^(−/−) hepatocytes dramatically suppresses the phenotype of steatohepatitis. In contrast, neurodegeneration due to expression and accumulation of polyglutamate expansion mutant proteins is greatly exacerbated by allelic loss of beclin1 and defective autophagy. Thus, while not intending to be bound by any theory of operation, autophagy is believed to be involved in limiting the buildup of misfolded, mutated proteins in p62-containing protein aggregates, which leads to cellular deterioration and disease.

Analogous to a wound-healing response, chronic tumor cell death in response to stress and induction of inflammation and cytokine production may provide a non-cell-autonomous mechanism by which tumorigenesis is promoted in autophagy-defective cells. Autophagy-defective tumor cells also display an elevated DNA damage response, gene amplification and chromosome instability in response to stress, suggesting that autophagy limits genome damage as a cell-autonomous mechanism of tumor suppression.

Therefore, while not intending to be bound by any theory of operation, stimulating autophagy may be involved in limiting disease progression, particularly neurodegeneration, liver disease, and also cancer, by facilitating the elimination of protein aggregates, damaged organelles, and the toxic consequences of their accumulation.

Autophagy has been identified also as a survival pathway in epithelial tumor cells that enables long-term survival to metabolic stress. Tumor cells with defined defects in autophagy accumulate p62-containing protein aggregates, DNA damage and die in response to stress, whereas those with intact autophagy can survive for weeks utilizing the autophagy survival pathway. Thus, autophagy appears to be required to prevent tumor cell damage and to maintain metabolism. Tumor cells can exploit this survival function to remain dormant only to reemerge under more favorable conditions. Interestingly, roughly half of human cancers may have impaired autophagy, either due to constitutive activation of the PI-3 kinase pathway or allelic loss of the essential autophagy gene beclin1, rendering them particularly susceptible to metabolic stress and autophagy inhibition.

Therefore, identification of the therapeutic means to inhibit the autophagy survival pathway in tumor cells would be advantageous. While not intending to be bound by any theory of operation, this may be of value as many therapeutics currently in use, such as kinase and angiogenesis inhibitors, inflict metabolic stress, which increases the dependency on autophagy for survival. Furthermore, tumor cells with impaired autophagy are particularly vulnerable to metabolic stress and further therapeutic suppression of autophagy may be able to exploit this vulnerability by promoting cell death by metabolic catastrophe or the failure to mitigate cell damage accumulation. Preclinical studies have been conducted using hydroxychloroquine to inhibit lysosome acidification and thereby autophagy in combination therapy. Specific inhibitors of the autophagy survival pathway in tumor cells are may be of great value in combination with agents such as angiogenesis and kinase inhibitors that promote metabolic stress.

Thus, the autophagy pathway represents fertile ground for novel therapeutic target identification for drug discovery for many diseases for both acute treatment and also disease prevention.

Accordingly, a need exists to identify nucleic acid sequences and their encoded proteins which are involved in modulation of the autophagy pathway.

BRIEF SUMMARY OF THE INVENTION

In certain aspects, the present invention relates to methods for identifying compounds that inhibit or stimulate the autophagy pathway.

Further aspects relate to methods for identifying individuals susceptible to or afflicted with a disease state associate with an autophagy pathway defect.

Additional aspects relate to devices for detecting the expression of autophagy-related genes.

Further aspects relate to kits for assaying expression of autophagy-related genes.

Other aspects are readily apparent from the following description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a cell-based shRNA screen for rescue of autophagy deficiency and p62 protein aggregate accumulation in metabolic stress: Screen for autophagy stimulators.

FIG. 2 illustrates representative images of shRNAs that modulate p62 aggregate elimination.

DETAILED DESCRIPTION OF THE INVENTION

The following definitions are provided to facilitate an understanding of the present invention:

A “polynucleotide,” “polynucleotide molecule” or “polynucleotide sequence” refers to a chain of nucleotides. It may refer to a DNA or RNA molecule, either single or double stranded and, if single stranded, the molecule of its complementary sequence in either linear or circular form. In some instances, the sequences will be fully complementary (no mismatches) when aligned. In other instances, there may be up to about a 30% mismatch in the sequences.

The term “oligonucleotide,” as used herein refers to sequences, primers and probes, and is defined as a nucleic acid molecule comprised of two or more ribo- or deoxyribonucleotides, preferably more than three. The exact size of the oligonucleotide will depend on various factors and on the particular application and use of the oligonucleotide.

The term “probe” as used herein refers to either a probe for a nucleic acid or a probe for a protein. When used in connection with nucleic acids, a “probe” refers to an oligonucleotide, polynucleotide or nucleic acid, either RNA or DNA, whether occurring naturally as in a purified restriction enzyme digest or produced synthetically, which is capable of annealing with or specifically hybridizing to a nucleic acid with sequences complementary to the probe. A probe may be either single stranded or double stranded. The exact length of the probe will depend upon many factors, including temperature, source of probe and method of use. The probes herein are selected to be “substantially” complementary to different strands of a particular target nucleic acid sequence. This means that the probes must be sufficiently complementary so as to be able to “specifically hybridize” or anneal with their respective target strands under a set of pre-determined conditions. Therefore, the probe sequence need not reflect the exact complementary sequence of the target. For example, a non complementary nucleotide fragment may be attached to the 5′ or 3′ end of the probe, with the remainder of the probe sequence being complementary to the target strand. Alternatively, non complementary bases or longer sequences can be interspersed into the probe, provided that the probe sequence has sufficient complementarity with the sequence of the target nucleic acid to anneal therewith specifically. When used in connection with a polypeptide, a “probe” is a protein- or polypeptide-binding substance or agent, capable of specifically binding a particular protein or protein fragment to the substantial exclusion of other proteins or protein fragments. Such binding agents may be any molecule to which the protein or peptide specifically binds, including DNA (for DNA binding proteins), antibodies (as described in greater detail herein), cell membrane receptors, peptides, cofactors, lectins, sugars, polysaccharides, cells, cell membranes, organelles and organellar membranes.

“Array” refers to an ordered arrangement of at least two probes on a substrate. At least one of the probes represents a control or standard, and the other, a probe of diagnostic or screening interest.

“Specific binding” refers to a special and precise interaction between two molecules which is dependent upon their structure, particularly their molecular side groups; for example, the intercalation of a regulatory protein into the major groove of a DNA molecule, the hydrogen bonding along the backbone between two single stranded nucleic acids, or the binding between an epitope of a protein and an agonist, antagonist, or antibody.

The term “specifically hybridize” refers to the association between two single stranded nucleic acid molecules of sufficiently complementary sequence to permit such hybridization under predetermined conditions generally used in the art (sometimes termed “substantially complementary”). For example, the term may refer to hybridization of a nucleic acid probe with a substantially complementary sequence contained within a single stranded DNA or RNA molecule according to an aspect of the invention, to the substantial exclusion of hybridization of the nucleic acid probe with single stranded nucleic acids of non-complementary sequence. When used in connection with the association between single stranded nucleic acid molecules, the term “specifically bind” may be used to indicate that the molecules “specifically hybridize” as described herein.

An “antibody” or “antibody molecule” is any immunoglobulin, including antibodies and fragments thereof, that binds to a specific antigen. The term includes polyclonal, monoclonal, chimeric, and bispecific antibodies. As used herein, antibody or antibody molecule contemplates both an intact immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule such as those portions known in the art as Fab, Fab′, F(ab′)2 and F(v).

“Sample” is used in its broadest sense as containing nucleic acids, proteins, antibodies, and the like. A sample may comprise, for example, a bodily fluid; the soluble fraction of a cell preparation, or an aliquot of media in which cells were grown; a chromosome, an organelle, or membrane isolated or extracted from a cell; genomic DNA, RNA, or cDNA in solution or bound to a substrate; a cell; a tissue or a tissue biopsy; a tissue print; a fingerprint, buccal cells, skin, or hair; and the like. Bodily fluids include, without limitation, whole blood, blood plasma, blood serum, sputum, urine, sweat, and lymph.

As used herein, the term “subject” or “patient” refers to both humans and animals, unless specified that the “subject” or “patient” is an animal or a human. An “individual” also refers to both humans and animals, unless specified that the “individual” is an animal or a human. Animal subjects are preferably vertebrates, and more preferably, mammals.

“Autophagy-associated” or “autophagy-related” as used herein with respect to a disease, condition or disorder refers to that which results from an increase or decrease in normal autophagy function and/or that which may be treated and/or prevented by modulation of the autophagy pathway. As used herein with respect to a biological molecule, such as, for example, a polynucleotide or polypeptide, “autophagy-associated” or “autophagy-related” refers to a molecule for which alteration of the expression, abundance and/or activity thereof leads to modulation of the autophagy pathway.

In certain embodiments, the present invention relates to the identification of genes whose expression modulates autophagy. These genes and their gene products may represent targets for therapeutic intervention in the autophagy pathway.

In accordance with aspects of the present invention, a number of polynucleotides comprising at least a fragment of a gene have been identified as representing molecules whose knockdown of expression modulates the function of the autophagy pathway. In certain aspects, knockdown of gene expression stimulates autophagy. In other aspects, knockdown of gene expression inhibits autophagy.

Embodiments of the invention include validation of the candidate genes and gene fragments described herein using known techniques for in vitro and in vivo analysis.

In accordance with various aspects of the present invention, combinations, compositions, devices and kits are provided that may be used in the practice of methods provided according to certain embodiments of the invention.

Certain embodiments relate to methods of detection of alterations in the autophagy pathway. Certain of these methods may be used to detect conditions in which autophagy is reduced. Certain of these methods may be used to detect conditions in which autophagy is increased.

In accordance with certain aspects of the invention, a combination is provided comprising a plurality of polynucleotide molecules wherein the polynucleotide molecules encode gene products associated with modulation of the autophagy pathway. In certain embodiments, the combination comprises a plurality of polynucleotides whose knockdown stimulates autophagy. In certain embodiments, the plurality of polynucleotide molecules comprise two or more molecules identified in Table 3 or fragments thereof.

In certain embodiments, the combination comprises a plurality of polynucleotides whose knockdown inhibits autophagy. In certain embodiments, the plurality of polynucleotide molecules comprise two or more molecules identified in Table 4 or fragments thereof.

An embodiment of the invention provides a method for identifying compounds that modulate autophagy-associated gene expression comprising: a) measuring standard expression by measuring transcription or translation products of one or more of the genes or gene fragments identified in Table 3 and/or 4, or fragments thereof, in a standard sample in the absence of a test compound; b) measuring test expression by measuring the transcription or translation products of one or more of the genes or gene fragments identified in Table 3 and/or 4, or fragments thereof, in a test sample in the presence of the test compound; and c) comparing the standard expression to the test expression, wherein a change in the test expression compared to the standard expression is indicative of an effect of the test compound on the expression of genes whose expression modulates the autophagy pathway. In certain embodiments, a plurality of two or more of the genes or gene fragments identified in Table 3 and/or 4, or fragments thereof are used.

One embodiment of the invention provides a method for identifying compounds that inhibit or stimulate the autophagy pathway for treatment of a disease state associated with an autophagy pathway defect, comprising measuring the effect of one or more test compounds on the inhibition or stimulation of a product of one or more of the genes or gene fragments identified in Table 3 or Table 4.

An embodiment provides a method for the detection of differential expression of autophagy-associated polypeptides in a sample, comprising the steps of: a) reacting protein binding molecules with polypeptides of the sample, thereby allowing specific binding to occur, wherein the polypeptides bound by the protein-binding molecules comprise one or more polypeptides encoded by the genes or gene fragments identified in Table 3 and/or Table 4 or fragments thereof; b) detecting specific binding; and c) comparing the specific binding in the sample with that of a standard, wherein differences between the standard and sample specific binding indicate differential expression of polypeptides in the sample. In certain embodiments, the protein-binding molecules are directed to polypeptides comprising a plurality of two or more polypeptides encoded by the genes or gene fragments identified in Table 3 and/or Table 4 or fragments thereof.

Another embodiment provides a method for the detection of differential expression of autophagy-associated nucleic acids in a sample, comprising the steps of: a) hybridizing polynucleotides comprising one or more molecules identified in Table 3 and/or Table 4 or fragments thereof with nucleic acids of the sample, thereby forming one or more hybridization complexes; b) detecting the hybridization complexes; and c) comparing the hybridization complexes with those of a standard, wherein differences between the standard and sample hybridization complexes indicate differential expression of nucleic acids in the sample. In certain embodiments, the polynucleotides comprise a plurality of two or more molecules identified in Table 3 and/or Table 4 or fragments thereof.

Another embodiment comprises a composition of matter comprising one or more probes for detecting expression of autophagy-associated genes, wherein the probes comprise one or more of: a) nucleic acid molecules that specifically hybridize to one or more of the genes or gene fragments identified in Table 3 and/or Table 4, or fragments thereof; or b) polypeptide binding agents that specifically bind to polypeptides produced by expression of one or more nucleic acid molecules comprising sequences selected from one or more of genes or gene fragments identified in Table 3 and/or Table 4, or fragments thereof. In certain embodiments, the composition of matter comprises a collection of two or more probes.

Another embodiment provides a device for detecting expression of a plurality of autophagy-related genes, comprising a substrate to which is affixed, at known locations, a plurality of probes, wherein the probes comprise: a) a plurality of oligonucleotides or polynucleotides, each of which specifically hybridizes to a different sequence selected from any of the sequences identified in Table 3 and/or Table 4 or fragments thereof; or b) a plurality of polypeptide binding agents, each of which specifically binds to a different polypeptide or fragment thereof produced by expression of a nucleic acid molecule comprising a sequence selected from the genes or gene fragments comprising any of the sequences identified in Table 3 and/or Table 4 or fragments thereof.

In certain embodiments, a device is provided for detecting the expression of a plurality of autophagy-related genes associated with an autophagy pathway defect, said device comprising a substrate to which is affixed at known locations a plurality of probes, wherein the probes comprise:

-   -   a) a plurality of oligonucleotides or polynucleotides, each of         which specifically binds to a different sequence selected from         any of the sequences identified in Table 3 or Table 4 or         fragments thereof; or     -   b) a plurality of polypeptide binding agents, each of which         specifically binds to a different polypeptide or fragment         thereof produced by expression of a gene or gene fragment         comprising any of the sequences identified in Table 3 or Table 4         or fragments thereof.

Another embodiment provides a method for measuring the effect of a test compound on expression of an autophagy-associated gene, wherein the gene is selected from the group consisting of the genes or gene fragments identified in Table 3 and/or 4, the method comprising measuring production of transcription or translation products produced by expression of the gene or gene fragment in the presence or absence of the test compound, wherein a change in the production of transcription or translation products in the presence of the test compound is indicative of an effect of the test compound on expression of the gene or gene fragment.

In an embodiment, the gene expression is measured by providing a DNA construct comprising a reporter gene coding sequence operably linked to transcription regulatory sequences of the autophagy-associated gene, and measuring formation of a reporter gene product in the presence or absence of the test compound.

Another embodiment provides a kit for assaying the expression of autophagy-related genes, comprising at least one container comprising a collection of two or more probes, wherein the probes comprise: a) oligonucleotides or polynucleotides that specifically hybridize to two or more genes or gene fragments comprising any of the sequences identified in Table 3 and/or Table 4, or fragments thereof; or b) polypeptide binding agents that specifically bind to polypeptides produced by expression of two or more genes or gene fragments comprising any of the sequences identified in Table 3 and/or Table 4, or fragments thereof. The kit preferably comprises instructions for performing an assay of gene expression.

In certain embodiments, the invention provides a kit for assaying the expression of autophagy-related genes associated with an autophagy pathway defect, comprising at least one container and a collection of two or more probes, wherein the probes comprise:

-   -   a) oligonucleotides or polynucleotides that specifically bind to         two or more genes or gene fragments comprising any of the         sequences identified in Table 3 or Table 4, or fragments         thereof; or     -   b) polypeptide binding agents that specifically bind to         polypeptides produced by expression of two or more genes or gene         fragments comprising any of the sequences identified in Table 3         or Table 4, or fragments thereof. The kit preferably comprises         instructions for performing an assay of gene expression.

The invention provides, in certain embodiments, methods for identifying compounds that are useful in modulating the autophagy pathway. Preferably, the methods include contacting at least one polypeptide encoded by the genes and/or gene fragments identified in Table 3 and/or Table 4 with a test substance and determining whether the test substance binds to the polypeptide. Further, in certain embodiments of the invention, a test substance may be determined to stimulate or inhibit the biological activity of the relevant gene product comprising at least one polypeptide encoded by the genes and/or gene fragments identified in Table 3 and/or Table 4 and thereby be identified as a compound useful for the modulation of the autophagy pathway. Such assays may, in certain embodiments, be performed in vitro and may, in certain embodiments, be performed in a cell-based assay. In some embodiments, substances identified as modulating expression or biological activity in vitro may be further tested in vivo to confirm relevant and effective activity.

Test substances or compounds contemplated by aspects of the invention include compounds from chemical libraries, including natural products and/or synthetic products from combinatorial chemical synthesis. Such substances may include, without limitation, polypeptides, oligonucleotides, polynucleotides, or organic molecules.

In a further embodiment is provided a method of modulating autophagy-associated gene expression in a cell by administering an effective amount of a composition under appropriate conditions to affect the expression of at least one gene associated with autophagy having a sequence selected from the sequences identified in Table 3 and/or Table 4, or fragments thereof.

In preferred embodiments, the composition comprises an inhibitor of gene expression. The inhibitor of gene expression may be selected from molecules including, but not limited to, an antisense RNA, a morpholino polynucleotide, and an interfering RNA (RNAi).

According to a still further aspect of the invention, there is provided a genetically-modified non-human animal that has been transformed to express higher, lower or absent levels of a protein according to any one of the aspects of the invention described herein. Preferably, said genetically-modified animal is a transgenic or knockout animal. Preferably, the genetically-modified animal is a rodent, most preferably a mouse.

An embodiment of the invention also provides a method for screening for a substance effective to treat an autophagy-associated disease condition, by contacting a non-human genetically-modified animal as described above with a candidate substance and determining the effect of the substance on the physiological state of the animal.

Certain embodiments of the invention provide methods and kits for diagnosis of, determining susceptibility to and/or developing a prognosis for an autophagy-associated disease state in a subject. In certain aspects, these may involve tests on subject samples. In certain embodiments, these may be nucleic acid based tests or polypeptide-based tests. In some embodiments, the method or kit may include probes that bind to at least one polynucleotide encoding an autophagy-associated polypeptode. In some embodiments, the a plurality of two or more probes may be used. In some embodiments, the method or kit may include polypeptide binding agents that bind to at least one autophagy-associated polypeptide. In some embodiments, a plurality of two or more polypeptide binding agents may be used. In certain embodiments, the polypeptide-binding agent comprises antibodies and/or antigen-binding portions of an antibody that specifically binds to one or more autophagy-associated polypeptides. Preferably, the autophagy-associated polypeptides are encoded by the gene or gene fragments identified in Table 3 and/or Table 4.

One embodiment provides a method for identifying individuals susceptible to or afflicted with a disease state associated with an autophagy pathway defect, comprising testing a biological sample from an individual for a characteristic of one or more polypeptides produced by expression of one or more of the genes or gene fragments identified in Table 3 or Table 4 that is indicative of said disease state, wherein said characteristic is selected from the presence of at least one of said polypeptides, the absence of at least one of said polypeptides, an elevated level of at least one of said polypeptides, a reduced level of at least one of said polypeptides and, for two or more of said polypeptides, combinations thereof.

An embodiment provides a method to diagnose or develop a prognosis for an autophagy-related disease in a subject, the method comprising: a) obtaining a sample from the subject; b) measuring in the sample the production of transcription or translation products produced by the expression of one or more autophagy-associated genes or gene fragments comprising any of the sequences identified in Table 3 and/or Table 4, or fragments thereof; c) comparing the transcription or translation products of the sample with that of a standard, wherein a difference in the expression of any of the autophagy-associated genes or gene fragments is indicative of autophagy-related disease.

In one embodiment is provided a kit for the diagnosis of an autophagy-associated disease in a subject comprising polynucleotide probes that specifically bind to one or more autophagy-associated polynucleotides or a fragment thereof. Preferably the autophagy-associated polynucleotides or fragments thereof are selected from the polynucleotide sequences identified in Table 3 and/or Table 4 or fragments thereof. In certain embodiments, the kit comprises a plurality of two or more polynucleotide probes that specifically bind to polynucleotide sequences identified in Table 3 and/or Table 4 or fragments thereof. Preferably, the kit comprises also instructions for use.

The invention also provides kits for diagnosis of autophagy-associated conditions from patient samples that may be nucleic acid based tests or polypeptide-based tests. In some embodiments, the kit contains at least one polynucleotide that binds to a polynucleotide encoding an autophagy-related gene product. In some embodiments, the kit contains, preferably in separate containers, a plurality of probes to detect two or more polynucleotides encoding one or more autophagy-associated gene products. In preferred embodiments, the gene products are encoded by one or more of the genes or gene fragments identified in Table 3 and/or Table 4. In other embodiments, the kit contains at least one polypeptide binding agent that specifically binds to at least one autophagy-associated polypeptide. In some embodiments, the kit contains, preferably in separate containers, a plurality of polypeptide binding agents (or mixtures thereof) to detect one or more autophagy-associated polypeptide. In certain embodiments, the polypeptide binding agent may be an antibody or antigen-binding portion of an antibody. In certain embodiments, the autophagy-associated polypeptides include at least one polypeptide encoded by the genes or gene fragments identified in Table 3 and/or Table 4. In certain embodiments, the autophagy-associated polypeptides identified by the kit include a plurality of two or more polypeptides encoded by the genes or gene fragments identified in Table 3 and/or Table 4. In certain embodiments, the kits may also include instructions for use.

In certain embodiments, methods according to the invention may be used for high-throughput screening assays.

In certain embodiments, methods and kits useful in the methods of the invention may utilize nucleic acid, antibody and/or polypeptide arrays.

Using a cell-based loss-of function screen, the present inventors have identified candidate genes whose expression is involved in the autophagy pathway. In particular, the screen has been used to identify genes whose knockdown stimulates autophagy. Results from this screen are shown in Table 1. The screen has also been used to identify genes whose knockdown inhibits autophagy. Results from this screen are shown in Table 2.

A high-efficiency delivery method that enables stable long-term gene suppression in a broad range of cell types is virus-mediated integration of an RNAi expression cassette. After integration, the cassette produces a short dsRNA molecule, usually in the form of a hairpin structure, a short or small hairpin RNA (shRNA), which is processed into active small interfering RNA (siRNA). Although many types of viruses are suitable for this purpose, lentiviral vectors generate viruses of both high titer and broad tropism, permitting the infection of both dividing and nondividing cells. Lentiviral shRNA libraries for mouse gene clones were utilized that allow gene silencing in most dividing and nondividing cell types.

An image based, arrayed shRNA screen was employed. Lentiviral shRNA libraries developed by the RNA Consortium (TRC) at the Broad Institute were used in a cell-based screen. The screens utilized the publicly available kinase and vesicle trafficking lentiviral library subsets at the Broad Institute, as well as a custom library containing shRNAs targeting mouse GTPases. Lentiviruses are high-titer, individual clones with representation of at least five independent hairpins for each target gene supplied in a high-throughput format (Root, D. E., et al. (2006), Nature Methods 3, 715-719.) Fluorescence image analysis was used to capture the data. Gene were identified that were shown to promote or suppress autophagy (bimodal analysis).

The high content arrayed shRNA screen used to identify autophagy modulators utilized autophagy defective beclin1^(+/−) iBMK cells stably expressing the autophagy substrate EGFP-p62 (Mathew, R., Karantza-Wadsworth, V., and White, E. (2009) Methods Enzymol 453, 53-81; Mathew, R., et al. (2009) Cell 137, 1062-1075; and Mathew, R., et al. (2007) Genes Dev 21, 1367-1381). p62 accumulates and aggregates in response to metabolic stress and requires autophagy for degradation. p62 also accumulates in degenerative neuronal and liver diseases and in autophagy-defective mouse tissues, beclin1^(+/−) and atg5^(−/−) iBMK cells, and tumors. Genes were identified whose inactivation compensates for defective autophagy and restores p62 protein turnover. Since the image analysis captured every hairpin's p62 aggregation score (EGFP-p62 intensity divided by the nuclei in the field) it was also possible to identify genes whose inactivation lead to further accumulation of p62 aggregates, predicted to be autophagy inhibitors (FIG. 2). This was possible because the cell line employed in this screen is autophagy impaired rather than fully autophagy defective. Therefore, the disposition of p62 in the test cells serves as readout for both autophagy promotion (p62 degradation, low p62) and inhibition (autophagy inhibition, high p62) and is the basis for the identification of autophagy modulators in cell-based screens.

The shRNA libraries were screened using autophagy-impaired test cells expressing a marker of protein aggregation, subjecting the test cell to metabolic stress, and performing analysis on the test cell to determine the level of the marker. The marker of protein aggregation is a p62 protein linked to enhanced green fluorescent protein (EGFP) label. Image analysis is performed to determine the level of p62 aggregates in cells. The level of the marker found in p62 aggregates in the test cell is compared with that of a control cell. A lower level of p62 aggregates comprising the marker in the test cell compared to that demonstrated by the control cell demonstrates the rescue of the impairment in p62 clearance, indicating the lowered expression of a gene whose knockdown stimulates autophagy. A greater level of p62 aggregates in a test cell compared to that of a control cell demonstrates suppression of p62 clearance, indicating the knockdown of a gene whose lowered level of expression leads to inhibition of autophagy.

The cell-based screen utilized autophagy-deficient beclin1^(+/−) immortalized baby mouse kidney (iBMK) cells stably expressing EGFP-p62. p62 accumulates and aggregates in response to metabolic stress and requires autophagy for degradation. FIG. 1 illustrates a cell-based shRNA screen for rescue of autophagy deficiency and p62 protein aggregate accumulation in metabolic stress and therefore represents a screen for autophagy stimulators. The autophagy-deficient beclin1^(+/−) iBMK cell line stably expressing EGFP-p62 accumulates p62-containing protein aggregates under stress, which fail to be cleared following recovery. Those shRNAs that facilitate p62 aggregate clearance, compensating for defective autophagy, are identified. The autophagy wild type beclin1^(+/+) iBMK cell line stably expressing EGFP-p62 that effectively clears p62 aggregates following stress is used as a positive control.

FIG. 2 illustrates representative images of cells contacted with shRNAs that modulate p62 aggregate elimination. The screen is designed to identify genes whose loss results in restoration of autophagy (autophagy stimulators), manifested by successful clearance of p62 aggregates following a time course of stress and recovery. mTOR, a master negative regulator of autophagy, is shown here as an example of a gene whose loss restores autophagy and clearance of p62. Alternatively, loss of some genes is predicted to further inhibit autophagy (autophagy inhibitors). Loss of Ikbkb, a known autophagy promoter (Criollo, A, et al. EMBO J 29, 619-631), results in marked accumulation of p62. This accumulation is greater than observed in cells infected with an shRNA targeting luciferase.

The shRNAs shown to promote p62 elimination (autophagy stimulators) identify potential targets for drug discovery efforts for development of modulators of autophagy, including autophagy inhibitors. While not intending to be bound by any theory of operation, autophagy inhibitors are potentially useful as anti-cancer therapeutics by promoting cancer cell death.

The shRNAs shown to enhance p62 accumulation (autophagy inhibitors) identify potential targets for drug discovery efforts for development of modulators of autophagy, including autophagy stimulators. While not intending to be bound by any theory of operation, autophagy stimulators are potentially useful in preventing or delaying disease manifestation in the setting of cancer, neurodegenerative conditions, Crohn's disease, liver disease, aging and inflammatory diseases and in combating infections.

EXAMPLES

The following examples serve to more fully describe the manner of using the above-described invention. It is understood that these examples in no way serve to limit the true scope of this invention, but rather are presented for illustrative purposes.

Materials and Methods Cell Line

Murine kidney epithelial cells were isolated from beclin1^(+/−) mice and immortalized with dominant negative p53 and EIA as described previously (Degenhardt, K., and White, E. (2006). Clin Cancer Res 12, 5298-5304; Mathew, R., Degenhardt, K., Haramaty, L., Karp, C. M., and White, E. (2008) Methods Enzymol 446, 77-106.). The cells were subsequently engineered to overexpress Bcl-2 and eGFP-p62. Thus, these cells, known as 3BC2 EGFP-P62, contain an autophagy defect, are apoptotically impaired, and stably express EGFP-P62. (Mathew, R., et al. (2009). Cell 137, 1062-1075.).

Screening Protocol for Beclin^(+/−) eGFP-P62

Cells were plated into black barcoded 384 well plates (Corning 8793BC) at a density of 700 cells/well by the Biotek microfill and allowed to attach overnight. Infection and media changes for plates were achieved by use of two robotic liquid handlers at the Broad Institute, the Perkin Elmer Janus and EP3. Each viral plate was used to infect four target plates. Each virus plate contained 20 control hairpins (targeting either RFP, luciferase, or EGFP) in addition to wells containing no virus. Two hairpins targeting p62 were spiked into each plate at the time of infection to ensure that positive and negative controls were present on all plates. Immediately prior to infection, media was changed with the Janus robot (Perkin Elmer) to DMEM containing 8 ug/mL polybrene. The Perkin Elmer EP3 robot was used to add 6 ul of virus to each well. Cells were spin infected (2250 rpm 30 mins, 30° C.) in the presence of 6 ul of virus and Bug/ml of polybrene before returning to the 37 C incubator. Virus and polybrene containing media was removed 4 hours post infection and cells were incubated in normal growth media overnight (DMEM high glucose, 10% FBS, 1% penicillin streptomycin (PS)). Twenty four hours post infection, media was changed with the Janus to DMEM containing 3 ug/mL puromycin (for the three puro plus plates) or DMEM alone (for the puro minus replicate). Selection was allowed to continue for 72 hrs.

The assay employed in this screen is predicated on the ability of autophagy competent cells to successfully eliminate p62 aggregates that accumulate during metabolic stress during a recovery phase during which time oxygen and glucose are restored. Optimization experiments were conducted comparing the ability of autophagy competent beclin1^(+/+)-EGFP-p62 cells (WB3-EGFP-p62) and autophagy deficient beclin1^(+/−)-EGFP-p62 cells (3Bc2-EGFP-p62) to eliminate p62 aggregates during various time courses of metabolic stress (1% oxygen, glucose deprivation) and recovery within the setting of 384 well plates post infection and selection with puromycin. 7.5 hours of metabolic stress followed by 18 hours of recovery in high glucose DMEM 10% FBS was optimal, and these conditions were chosen for the large-scale screen.

Following puromycin selection, media containing DMEM high glucose was removed, and cells were washed twice in ischemia media (DMEM containing no glucose, 10% FBS, 1% PS) to remove residual glucose in wells prior to transfer into a hypoxia incubator set to 1% oxygen for 7.5 hours. They were then transferred to an incubator which could lower ambient oxygen levels to 1% by virtue of its attachment to a nitrogen tank. Cells stayed in this 1% oxygen, no glucose conditions, referred to as metabolic stress, for 7.5 h.

At the conclusion of the metabolic stress, normal growth media (DMEM high glucose 10% FBS, 1% PS) was added to the plates and the cells were allowed to recover overnight at 37° C. 18 hours post recovery, media was removed from plates, and cells were fixed by addition of 4% paraformaldehyde/PBS for 10 mins at RT.

Nuclei were visualized by inclusion of Hoechst 33342 at a dilution of 1:10,000 in the fixation solution. Plates were washed 3× with the ELx405 automated plate washer (Biotek). 80 ul of filtered PBS was left in each well at the end of washing to allow for evaporation during imaging.

Plates were imaged on the Arrayscan VTI (Thermo Scientific) housed within the Genome technology Core of the Whitehead Institute using a modified version of the Cellomics compartmental analysis bioapplication. Nuclei were visualized in channel 1. EGFP-p62 aggregates were visualized in channel 2. Nine images per channel were captured for each field, with an autofocus field interval of 3. MEAN_valid object count channel 1 represents the mean nuclear count within the field. MEAN_ring spot average integrated intensity channel 2 represents the mean intensity of the p62 aggregates in the field. To properly identify the p62 aggregates the following settings were employed: Spot kernel radius: 10, ring distance from nucleus: 0, ring width: 10 pixels. Data was exported to Excel for further analysis. Data quality (batch-to-batch variation, similarity of replicates) was examined with Spotfire decision software and RNAeyes, in house software developed by The RNAi Consortium (TRC) of the Broad Institute.

A p62 aggregate score equal to Mean Ring Spot total intensity/Mean nuclei was calculated for each well. Viral infections were done in quadruplicate, with three plates receiving puromycin, one not. A comparison of the nuclei counts from the puro+/puro− plates allowed calculation of the infection efficiency of each hairpin. Hairpins with less than 1500 nuclei per well or those that had an infection efficiency less than 25% were omitted from subsequent analysis. A robust Z-score, a standard metric for high throughput assays (Birmingham A., et al. (2009) Nat Methods 6, 569-575), was calculated for each well. The three puromycin selected replicates were averaged, and this value was used for further analysis.

The in-house Gene-E software ranked genes at both a hairpin and a gene level. Attached to this application are candidate results (‘hits’) from either end of our analysis: those that resulted in profound elimination of p62, predicted to be autophagy inducers (Table 1), and those that resulted in profound accumulation of p62, predicted to be autophagy inhibitors (Table 2). These tables represent the weighted sum ranking of the data. In this metric, 75% of the score is based on the robust z-score of the second best hairpin for a given gene, while the other 25% of the score is based on the rank of the robust z-score of the best hairpin. Similar data was obtained when three other analysis measures were employed: cut-off based on a given standard deviation from controls, second best ranking, or RNA Interference Gene Enrichment Rank (RIGER) analysis based on the KS statistic as described previously (Luo B., et al. (2008) Proc Natl Acad Sci USA 105, 20380-20385).

A subset of viral plates were re-screened to ensure reproducibility of the assay and analyses.

Example 1

Table 1 shows results of a screen that led to elimination of p62.

TABLE 1 Symbol GeneID Gene Rank Mtor 56717 1 Tssk3 58864 2 Pik3c3 225326 3 Cask 12361 4 Lrguk 74354 5 GeneID: 218456 218456 6 Rab9 56382 7 GeneID: 381390 381390 8 Gm4922 237300 9 Cdk8 264064 10 Ephb1 270190 11 Prkaca 18747 12 Kpna2 16647 13 Pldn 18457 14 Scfd1 76983 15 Ripk3 56532 16 Trib3 228775 17 Vapa 30960 18 Trrap 100683 19 Mpp3 13384 20 GeneID: 381082 381082 21 Mapk14 26416 22 Adk 11534 23 Ern1 78943 24 Hip1r 29816 25 Nek5 330721 26 Alpk3 116904 27 4932415M13Rik 211496 28 Vps33b 233405 29 1810024B03Rik 329509 30 Chmp1a 234852 31 Atp6v0a1 11975 32 Ddr2 18214 33 Cdk6 12571 34 Stxbp3a 20912 35 Map4k3 225028 36 Egfr 13649 37 Tpr 108989 38 Tlk2 24086 39 Rhoh 74734 40 Sar1a 20224 41 Vta1 66201 42 Rab34 19376 43 Brdt 114642 44 GFP −10 45 GeneID: 384481 384481 46 Dgka 13139 47 Rabl2a 68708 48 Snx10 71982 49 Rhoa 11848 50 Map3k11 26403 51 Gm5374 385049 52 D1g4 13385 53 Rab7l1 226422 54 Vamp8 22320 55 N4bp2 333789 56 Arf3 11842 57 GeneID: 381309 381309 58 Plk2 20620 59 Cpne3 70568 60 Hip1 215114 61 Musk 18198 62 Rab39b 67790 63 Akt1 11651 64 Arhgap24 231532 65 Eif2ak1 15467 66 Pfkfb4 270198 67 Txndc3 73412 68 Pim1 18712 69 5730410E15Rik 319613 70 1190002A17Rik 68870 71 Gvin1 74558 72 Pank4 269614 73 Bmpr1a 12166 74 Grk4 14772 75 Pip5k1a 18720 76 Prkcz 18762 77 Nek3 23954 78 Pgk1 18655 79 Kras 16653 80 Tssk6 83984 81 Rab2a 59021 82 Mertk 17289 83 Ap1m2 11768 84 Snx2 67804 85 Ilk 16202 86 Dgkk 331374 87 Csnk1d 104318 88 Rps6kb2 58988 89 Map3k4 26407 90 Ca1m1 12313 91 Trim24 21848 92 Fyn 14360 93 Sh3bp5 24056 94 Fn3k 63828 95 Ippk 75678 96 Tspan1 66805 97 Cd81 12520 98 Met 17295 99 Pdgfra 18595 100 Vrk2 69922 101 Gem 14579 102 Camkk1 55984 103 Pfkl 18641 104 Stx8 55943 105 Tspan9 109246 106 Stx17 67727 107 Tm4sf1 17112 108 Chmp4c 66371 109 Tbk1 56480 110 Dgkh 380921 111 Ephb4 13846 112 Rhof 23912 113 Pik3cd 18707 114 Mark1 226778 115 Tspan2 70747 116 Dync1li1 235661 117 Trim27 19720 118 Aurkc 20871 119 Itpkb 320404 120 Cav1 12389 121 Bub1 12235 122 Rap1b 215449 123 Mapk10 26414 124 Mapk8 26419 125 Rab24 19336 126 Kdr 16542 127 Rab2b 76338 128 Irak3 73914 129 Map3k8 26410 130 Csnk1a1 93687 131 Rhobtb1 69288 132 Mast2 17776 133 Raf1 110157 134 Arl11 219144 135 Dgkq 110524 136 Arfrp1 76688 137 Mknk2 17347 138 Erbb3 13867 139 Rheb 19744 140 Clk4 12750 141 Map2k1 26395 142 Sbk1 104175 143 Clk3 102414 144 Irak1 16179 145 Pik3cb 74769 146 Map3k12 26404 147 Tk1 21877 148 Aatk 11302 149 Fastkd2 75619 150

TABLE 3 GeneID RefSeq Species Description 270198 NM_001039217 MUS 6-PHOSPHOFRUCTO-2-KINASE/FRUCTOSE- MUSCULUS 2,6-BIPHOSPHATASE 4 270198 NM_001039215 MUS 6-PHOSPHOFRUCTO-2-KINASE/FRUCTOSE- MUSCULUS 2,6-BIPHOSPHATASE 4 270198 NM_001039216 MUS 6-PHOSPHOFRUCTO-2-KINASE/FRUCTOSE- MUSCULUS 2,6-BIPHOSPHATASE 4 270198 NM_173019 MUS 6-PHOSPHOFRUCTO-2-KINASE/FRUCTOSE- MUSCULUS 2,6-BIPHOSPHATASE 4 11768 NM_009678 MUS ADAPTOR PROTEIN COMPLEX AP-1, MU 2 MUSCULUS SUBUNIT 11534 NM_134079 MUS ADENOSINE KINASE MUSCULUS 11842 NM_007478 MUS ADP-RIBOSYLATION FACTOR 3 MUSCULUS 76688 NM_029702 MUS ADP-RIBOSYLATION FACTOR RELATED MUSCULUS PROTEIN 1 219144 NM_177337 MUS ADP-RIBOSYLATION FACTOR-LIKE 11 MUSCULUS 116904 NM_054085 MUS ALPHA-KINASE 3 MUSCULUS 11302 NM_007377 MUS APOPTOSIS-ASSOCIATED TYROSINE MUSCULUS KINASE 11975 NM_016920 MUS ATPASE, H+ TRANSPORTING, LYSOSOMAL MUSCULUS V0 SUBUNIT A1 20871 NM_020572 MUS AURORA KINASE C MUSCULUS 333789 NM_001024917 MUS BCL3 BINDING PROTEIN MUSCULUS 12166 NM_009758 MUS BONE MORPHOGENETIC PROTEIN MUSCULUS RECEPTOR, TYPE 1A 114642 NM_054054 MUS BROMODOMAIN, TESTIS-SPECIFIC MUSCULUS 12235 NM_009772 MUS BUDDING UNINHIBITED BY MUSCULUS BENZIMIDAZOLES 1 HOMOLOG (S. CEREVISIAE) 17289 NM_008587 MUS C-MER PROTO-ONCOGENE TYROSINE MUSCULUS KINASE 55984 NM_018883 MUS CALCIUM/CALMODULIN-DEPENDENT MUSCULUS PROTEIN KINASE KINASE 1, ALPHA 12361 NM_009806 MUS CALCIUM/CALMODULIN-DEPENDENT MUSCULUS SERINE PROTEIN KINASE (MAGUK FAMILY) 12313 NM_007589 MUS CALMODULIN 1 MUSCULUS 12313 NM_007590 MUS CALMODULIN 1 MUSCULUS 12313 NM_009790 MUS CALMODULIN 1 MUSCULUS 104318 NM_027874 MUS CASEIN KINASE 1, DELTA MUSCULUS 104318 NM_139059 MUS CASEIN KINASE 1, DELTA MUSCULUS 93687 NM_146087 MUS CASEIN KINASE I-ALPHA MUSCULUS 12389 NM_007616 MUS CAVEOLIN, CAVEOLAE PROTEIN 1 MUSCULUS 12520 NM_133655 MUS SP. CD 81 ANTIGEN 12520 NM_133655 MUS CD 81 ANTIGEN MUSCULUS 12750 NM_007714 MUS CDC LIKE KINASE 4 MUSCULUS 102414 NM_007713 MUS CDC-LIKE KINASE 3 MUSCULUS 70568 NM_027769 MUS COPINE III MUSCULUS 12571 NM_009873 MUS CYCLIN-DEPENDENT KINASE 6 MUSCULUS 264064 NM_181570 MUS CYCLIN-DEPENDENT KINASE 8 MUSCULUS 264064 NM_153599 MUS CYCLIN-DEPENDENT KINASE 8 MUSCULUS 331374 NM_177914 MUS DIACYLGLYCEROL KINASE KAPPA MUSCULUS 13139 NM_016811 MUS DIACYLGLYCEROL KINASE, ALPHA MUSCULUS 380921 NM_001081336 MUS DIACYLGLYCEROL KINASE, ETA XM_895030 MUSCULUS 380921 XM_902438 MUS DIACYLGLYCEROL KINASE, ETA MUSCULUS 380921 XM_484397 MUS DIACYLGLYCEROL KINASE, ETA MUSCULUS 380921 XM_916777 MUS DIACYLGLYCEROL KINASE, ETA MUSCULUS 380921 XM_924467 MUS DIACYLGLYCEROL KINASE, ETA MUSCULUS 110524 NM_199011 MUS DIACYLGLYCEROL KINASE, THETA MUSCULUS 18214 NM_022563 MUS DISCOIDIN DOMAIN RECEPTOR FAMILY, MUSCULUS MEMBER 2 13385 NM_007864 MUS DISCS, LARGE HOMOLOG 4 (DROSOPHILA) MUSCULUS 235661 NM_146229 MUS DYNEIN CYTOPLASMIC 1 LIGHT MUSCULUS INTERMEDIATE CHAIN 1 78943 NM_023913 MUS ENDOPLASMIC RETICULUM (ER) TO MUSCULUS NUCLEUS SIGNALLING 1 270190 NM_173447 MUS EPH RECEPTOR B1 MUSCULUS 13846 NM_010144 MUS EPH RECEPTOR B4 MUSCULUS 13649 NM_007912 MUS EPIDERMAL GROWTH FACTOR RECEPTOR MUSCULUS 13649 NM_207655 MUS EPIDERMAL GROWTH FACTOR RECEPTOR MUSCULUS 15467 NM_013557 MUS EUKARYOTIC TRANSLATION INITIATION MUSCULUS FACTOR 2 ALPHA KINASE 1 56717 NM_020009 MUS FK506 BINDING PROTEIN 12-RAPAMYCIN MUSCULUS ASSOCIATED PROTEIN 1 56717 NM_001039554 MUS FK506 BINDING PROTEIN 12-RAPAMYCIN MUSCULUS ASSOCIATED PROTEIN 1 63828 NM_001038699 MUS FRUCTOSAMINE 3 KINASE MUSCULUS 63828 NM_022014 MUS FRUCTOSAMINE 3 KINASE MUSCULUS 14360 NM_008054 MUS FYN PROTO-ONCOGENE MUSCULUS 14772 NM_019497 MUS G PROTEIN-COUPLED RECEPTOR KINASE MUSCULUS 2, GROUCHO GENE RELATED (DROSOPHILA) 14579 NM_010276 MUS GTP BINDING PROTEIN (GENE MUSCULUS OVEREXPRESSED IN SKELETAL MUSCLE) 74558 NM_001039160 MUS GTPASE, VERY LARGE INTERFERON MUSCULUS INDUCIBLE 1 74558 NM_029000 MUS GTPASE, VERY LARGE INTERFERON MUSCULUS INDUCIBLE 1 29816 NM_145070 MUS HUNTINGTIN INTERACTING PROTEIN 1 MUSCULUS RELATED 237300 NM_177706 MUS HYPOTHETICAL PROTEIN 4933423E17 MUSCULUS 228775 NM_175093 MUS INDUCED IN FATTY LIVER DYSTROPHY 2 MUSCULUS 228775 NM_144554 MUS INDUCED IN FATTY LIVER DYSTROPHY 2 MUSCULUS 75678 NM_199056 MUS INOSITOL 1,3,4,5,6-PENTAKISPHOSPHATE MUSCULUS 2-KINASE 320404 NM_001081175 MUS INOSITOL 1,4,5-TRISPHOSPHATE 3-KINASE B XM_205854 MUSCULUS 320404 XM_923874 MUS INOSITOL 1,4,5-TRISPHOSPHATE 3-KINASE B MUSCULUS 320404 XM_915655 MUS INOSITOL 1,4,5-TRISPHOSPHATE 3-KINASE B MUSCULUS 320404 XM_900404 MUS INOSITOL 1,4,5-TRISPHOSPHATE 3-KINASE B MUSCULUS 16202 NM_010562 MUS INTEGRIN LINKED KINASE MUSCULUS 16179 NM_008363 MUS INTERLEUKIN-1 RECEPTOR-ASSOCIATED MUSCULUS KINASE 1 73914 NM_028679 MUS INTERLEUKIN-1 RECEPTOR-ASSOCIATED MUSCULUS KINASE 3 16647 NM_010655 MUS KARYOPHERIN (IMPORTIN) ALPHA 2 MUSCULUS 16542 NM_010612 MUS SP. KINASE INSERT DOMAIN PROTEIN RECEPTOR 16542 NM_010612 MUS KINASE INSERT DOMAIN PROTEIN MUSCULUS RECEPTOR 17347 NM_021462 MUS MAP KINASE-INTERACTING MUSCULUS SERINE/THREONINE KINASE 2 226778 NM_145515 MUS MAP/MICROTUBULE AFFINITY- MUSCULUS REGULATING KINASE 1 13384 NM_007863 MUS MEMBRANE PROTEIN, PALMITOYLATED 3 MUSCULUS (MAGUK P55 SUBFAMILY MEMBER 3) 17295 NM_008591 MUS MET PROTO-ONCOGENE MUSCULUS 17776 NM_008641 MUS MICROTUBULE ASSOCIATED MUSCULUS SERINE/THREONINE KINASE 2 26414 NM_009158 MUS MITOGEN ACTIVATED PROTEIN KINASE MUSCULUS 10 26416 NM_011951 MUS MITOGEN ACTIVATED PROTEIN KINASE MUSCULUS 14 26419 NM_016700 MUS MITOGEN ACTIVATED PROTEIN KINASE 8 MUSCULUS 26395 NM_008927 MUS MITOGEN ACTIVATED PROTEIN KINASE MUSCULUS KINASE 1 26403 NM_022012 MUS MITOGEN ACTIVATED PROTEIN KINASE MUSCULUS KINASE KINASE 11 26404 NM_009582 MUS MITOGEN ACTIVATED PROTEIN KINASE MUSCULUS KINASE KINASE 12 26407 NM_011948 MUS MITOGEN ACTIVATED PROTEIN KINASE MUSCULUS KINASE KINASE 4 26410 NM_007746 MUS MITOGEN ACTIVATED PROTEIN KINASE MUSCULUS KINASE KINASE 8 18198 NM_001037128 MUS MUSCLE, SKELETAL, RECEPTOR MUSCULUS TYROSINE KINASE 18198 NM_010944 MUS MUSCLE, SKELETAL, RECEPTOR MUSCULUS TYROSINE KINASE 18198 NM_001037127 MUS MUSCLE, SKELETAL, RECEPTOR MUSCULUS TYROSINE KINASE 18198 NM_001037129 MUS MUSCLE, SKELETAL, RECEPTOR MUSCULUS TYROSINE KINASE 18198 NM_001037130 MUS MUSCLE, SKELETAL, RECEPTOR MUSCULUS TYROSINE KINASE 23954 NM_011848 MUS NIMA (NEVER IN MITOSIS GENE A)- MUSCULUS RELATED EXPRESSED KINASE 3 330721 NM_177898 MUS NIMA (NEVER IN MITOSIS GENE A)- MUSCULUS RELATED EXPRESSED KINASE 5 18457 NM_019788 MUS PALLIDIN MUSCULUS 269614 NM_172990 MUS PANTOTHENATE KINASE 4 MUSCULUS 18707 NM_001029837 MUS PHOSPHATIDYLINOSITOL 3-KINASE MUSCULUS CATALYTIC DELTA POLYPEPTIDE 18707 NM_008840 MUS PHOSPHATIDYLINOSITOL 3-KINASE MUSCULUS CATALYTIC DELTA POLYPEPTIDE 74769 NM_029094 MUS PHOSPHATIDYLINOSITOL 3-KINASE, MUSCULUS CATALYTIC, BETA POLYPEPTIDE 18720 NM_008847 MUS PHOSPHATIDYLINOSITOL-4-PHOSPHATE 5- MUSCULUS KINASE, TYPE 1 BETA 18641 NM_008826 MUS PHOSPHOFRUCTOKINASE, LIVER, B-TYPE MUSCULUS 18655 NM_008828 MUS PHOSPHOGLYCERATE KINASE 1 MUSCULUS 18655 XM_484116 MUS PHOSPHOGLYCERATE KINASE 1 MUSCULUS 18655 XM_485239 MUS PHOSPHOGLYCERATE KINASE 1 MUSCULUS 225326 NM_181414 MUS PHOSPHOINOSITIDE-3-KINASE, CLASS 3 MUSCULUS 18595 NM_011058 MUS PLATELET DERIVED GROWTH FACTOR MUSCULUS RECEPTOR, ALPHA POLYPEPTIDE 20620 NM_152804 MUS POLO-LIKE KINASE 2 (DROSOPHILA) MUSCULUS 234852 NM_145606 MUS PROCOLLAGEN (TYPE III) N- MUSCULUS ENDOPEPTIDASE 18762 NM_001039079 MUS PROTEIN KINASE C, ZETA MUSCULUS 18762 NM_008860 MUS PROTEIN KINASE C, ZETA MUSCULUS 18747 NM_008854 MUS PROTEIN KINASE, CAMP DEPENDENT, MUSCULUS CATALYTIC, ALPHA 18712 NM_008842 MUS PROVIRAL INTEGRATION SITE 1 MUSCULUS 68708 NM_026817 MUS RAB, MEMBER OF RAS ONCOGENE MUSCULUS FAMILY-LIKE 2A 59021 NM_021518 MUS RAB2, MEMBER RAS ONCOGENE FAMILY MUSCULUS 19336 NM_009000 MUS RAB24, MEMBER RAS ONCOGENE FAMILY MUSCULUS 76338 NM_172601 MUS RAB2B, MEMBER RAS ONCOGENE FAMILY MUSCULUS 19376 NM_033475 MUS SP. RAB34, MEMBER OF RAS ONCOGENE FAMILY 19376 NM_033475 MUS RAB34, MEMBER OF RAS ONCOGENE MUSCULUS FAMILY 67790 NM_175122 MUS RAB39B, MEMBER RAS ONCOGENE MUSCULUS FAMILY 226422 NM_144875 MUS RAB7, MEMBER RAS ONCOGENE FAMILY- MUSCULUS LIKE 1 56382 NM_019773 MUS RAB9, MEMBER RAS ONCOGENE FAMILY MUSCULUS 11848 NM_016802 MUS RAS HOMOLOG GENE FAMILY, MEMBER A MUSCULUS 23912 NM_175092 MUS RAS HOMOLOG GENE FAMILY, MEMBER F MUSCULUS 74734 NM_001081105 MUS RAS HOMOLOG GENE FAMILY, MEMBER H XM_132051 MUSCULUS 74734 XM_903893 MUS RAS HOMOLOG GENE FAMILY, MEMBER H MUSCULUS 74734 XM_903680 MUS RAS HOMOLOG GENE FAMILY, MEMBER H MUSCULUS 74734 XM_924029 MUS RAS HOMOLOG GENE FAMILY, MEMBER H MUSCULUS 74734 XM_622908 MUS RAS HOMOLOG GENE FAMILY, MEMBER H MUSCULUS 74734 XM_924031 MUS RAS HOMOLOG GENE FAMILY, MEMBER H MUSCULUS 74734 XM_915950 MUS RAS HOMOLOG GENE FAMILY, MEMBER H MUSCULUS 74734 XM_900704 MUS RAS HOMOLOG GENE FAMILY, MEMBER H MUSCULUS 74734 XM_132051 MUS RAS HOMOLOG GENE FAMILY, MEMBER H MUSCULUS 215449 NM_024457 MUS RAS RELATED PROTEIN 1B MUSCULUS 19744 NM_053075 MUS RAS-HOMOLOG ENRICHED IN BRAIN MUSCULUS 56532 NM_019955 MUS RECEPTOR-INTERACTING SERINE- MUSCULUS THREONINE KINASE 3 69288 NM_001081347 MUS RHO-RELATED BTB DOMAIN CONTAINING 1 XM_897555 MUSCULUS 69288 XM_897555 MUS RHO-RELATED BTB DOMAIN CONTAINING 1 MUSCULUS 69288 XM_125637 MUS RHO-RELATED BTB DOMAIN CONTAINING 1 MUSCULUS 69288 XM_920631 MUS RHO-RELATED BTB DOMAIN CONTAINING 1 MUSCULUS 69288 XM_920652 MUS RHO-RELATED BTB DOMAIN CONTAINING 1 MUSCULUS 69288 XM_897548 MUS RHO-RELATED BTB DOMAIN CONTAINING 1 MUSCULUS 69288 XM_907869 MUS RHO-RELATED BTB DOMAIN CONTAINING 1 MUSCULUS 69288 XM_897523 MUS RHO-RELATED BTB DOMAIN CONTAINING 1 MUSCULUS 69288 XM_920622 MUS RHO-RELATED BTB DOMAIN CONTAINING 1 MUSCULUS 69288 XM_897577 MUS RHO-RELATED BTB DOMAIN CONTAINING 1 MUSCULUS 69288 XM_920637 MUS RHO-RELATED BTB DOMAIN CONTAINING 1 MUSCULUS 69288 XM_920646 MUS RHO-RELATED BTB DOMAIN CONTAINING 1 MUSCULUS 69288 XM_897586 MUS RHO-RELATED BTB DOMAIN CONTAINING 1 MUSCULUS 69288 XM_920664 MUS RHO-RELATED BTB DOMAIN CONTAINING 1 MUSCULUS 69288 XM_887557 MUS RHO-RELATED BTB DOMAIN CONTAINING 1 MUSCULUS 69288 XM_920656 MUS RHO-RELATED BTB DOMAIN CONTAINING 1 MUSCULUS 58988 NM_021485 MUS RIBOSOMAL PROTEIN S6 KINASE, MUSCULUS POLYPEPTIDE 2 231532 NM_146161 MUS RIKEN CDNA 0610025G21 GENE MUSCULUS 231532 NM_029270 MUS RIKEN CDNA 0610025G21 GENE MUSCULUS 66201 NM_025418 MUS RIKEN CDNA 1110059P08 GENE MUSCULUS 68870 NM_001033874 MUS RIKEN CDNA 1190002A17 GENE MUSCULUS 329509 NM_198630 MUS RIKEN CDNA 1810024B03 GENE MUSCULUS 66371 NM_025519 MUS RIKEN CDNA 2310010I16 GENE MUSCULUS 75619 NM_172422 MUS RIKEN CDNA 2810421I24 GENE MUSCULUS 225028 NM_001081357 MUS RIKEN CDNA 4833416M01 GENE XM_898848 MUSCULUS 225028 XM_898825 MUS RIKEN CDNA 4833416M01 GENE MUSCULUS 225028 XM_898819 MUS RIKEN CDNA 4833416M01 GENE MUSCULUS 225028 XM_898848 MUS RIKEN CDNA 4833416M01 GENE MUSCULUS 225028 XM_898843 MUS RIKEN CDNA 4833416M01 GENE MUSCULUS 225028 XM_898830 MUS RIKEN CDNA 4833416M01 GENE MUSCULUS 225028 XM_898852 MUS RIKEN CDNA 4833416M01 GENE MUSCULUS 225028 XM_898838 MUS RIKEN CDNA 4833416M01 GENE MUSCULUS 74354 XM_910825 MUS RIKEN CDNA 4921528H16 GENE MUSCULUS 74354 XM_895665 MUS RIKEN CDNA 4921528H16 GENE MUSCULUS 74354 NM_028886 MUS LEUCINE-RICH REPEATS AND XM_133060 MUSCULUS GUANYLATE KINASE DOMAIN XM_910825 CONTAINING (LRGUK) 74354 XM_921792 MUS RIKEN CDNA 4921528H16 GENE MUSCULUS 211496 NM_177599 MUS RIKEN CDNA 4932415M13 GENE MUSCULUS 211496 NM_001037718 MUS RIKEN CDNA 4932415M13 GENE MUSCULUS 319613 NM_176998 MUS RIKEN CDNA 5730410E15 GENE MUSCULUS 319613 NM_001032727 MUS RIKEN CDNA 5730410E15 GENE MUSCULUS 319613 NM_178765 MUS RIKEN CDNA 5730410E15 GENE MUSCULUS 110157 NM_029780 MUS RIKEN CDNA 6430402F14 GENE MUSCULUS 215114 NM_146001 MUS RIKEN CDNA A930014B11 GENE MUSCULUS 20224 NM_009120 MUS SAR1 GENE HOMOLOG A (S. CEREVISIAE) MUSCULUS 76983 NM_029825 MUS SEC1 FAMILY DOMAIN CONTAINING 1 MUSCULUS 104175 NM_145587 MUS SH3-BINDING KINASE 1 MUSCULUS 24056 NM_011894 MUS SH3-DOMAIN BINDING PROTEIN 5 (BTK- MUSCULUS ASSOCIATED) 385049 XM_001473528 MUS SIMILAR TO RHO-ASSOCIATED COILED- XM_904204 MUSCULUS COIL FORMING KINASE 1 385049 XM_358017 MUS SIMILAR TO RHO-ASSOCIATED COILED- MUSCULUS COIL FORMING KINASE 1 381390 XM_485079 MUS SIMILAR TO SERINE/THREONINE KINASE MUSCULUS 381390 XM_355352 MUS GM14147 PREDICTED GENE 14147 MUSCULUS 71982 NM_028035 MUS SORTING NEXIN 10 MUSCULUS 67804 NM_026386 MUS SORTING NEXIN 2 MUSCULUS 67727 NM_026343 MUS SYNTAXIN 17 MUSCULUS 55943 NM_018768 MUS SYNTAXIN 8 MUSCULUS 20912 NM_011504 MUS SYNTAXIN BINDING PROTEIN 3A MUSCULUS 20912 NM_198326 MUS SYNTAXIN BINDING PROTEIN 3A MUSCULUS 56480 NM_019786 MUS TANK-BINDING KINASE 1 MUSCULUS 58864 NM_080442 MUS TESTIS-SPECIFIC SERINE KINASE 3 MUSCULUS 83984 NM_032004 MUS TESTIS-SPECIFIC SERINE KINASE 6 MUSCULUS 66805 NM_133681 MUS TETRASPANIN 1 MUSCULUS 70747 NM_027533 MUS TETRASPANIN 2 MUSCULUS 109246 NM_175414 MUS TETRASPANIN 9 MUSCULUS 73412 NM_181591 MUS THIOREDOXIN DOMAIN CONTAINING 3 MUSCULUS (SPERMATOZOA) 21877 NM_009387 MUS SP. THYMIDINE KINASE 1 21877 NM_009387 MUS THYMIDINE KINASE 1 MUSCULUS 11651 NM_009652 MUS THYMOMA VIRAL PROTO-ONCOGENE 1 MUSCULUS 24086 NM_011903 MUS TOUSLED-LIKE KINASE 2 (ARABIDOPSIS) MUSCULUS 100683 XM_891798 MUS TRANSFORMATION/TRANSCRIPTION MUSCULUS DOMAIN-ASSOCIATED PROTEIN 100683 XM_899747 MUS TRANSFORMATION/TRANSCRIPTION MUSCULUS DOMAIN-ASSOCIATED PROTEIN 100683 XM_918276 MUS TRANSFORMATION/TRANSCRIPTION MUSCULUS DOMAIN-ASSOCIATED PROTEIN 100683 NM_001081362 MUS TRANSFORMATION/TRANSCRIPTION XM_899741 MUSCULUS DOMAIN-ASSOCIATED PROTEIN 100683 XM_899763 MUS TRANSFORMATION/TRANSCRIPTION MUSCULUS DOMAIN-ASSOCIATED PROTEIN 100683 XM_917315 MUS TRANSFORMATION/TRANSCRIPTION MUSCULUS DOMAIN-ASSOCIATED PROTEIN 100683 XM_886427 MUS TRANSFORMATION/TRANSCRIPTION MUSCULUS DOMAIN-ASSOCIATED PROTEIN 100683 XM_899754 MUS TRANSFORMATION/TRANSCRIPTION MUSCULUS DOMAIN-ASSOCIATED PROTEIN 100683 XM_925613 MUS TRANSFORMATION/TRANSCRIPTION MUSCULUS DOMAIN-ASSOCIATED PROTEIN 100683 XM_925614 MUS TRANSFORMATION/TRANSCRIPTION MUSCULUS DOMAIN-ASSOCIATED PROTEIN 100683 XM_899771 MUS TRANSFORMATION/TRANSCRIPTION MUSCULUS DOMAIN-ASSOCIATED PROTEIN 100683 XM_918275 MUS TRANSFORMATION/TRANSCRIPTION MUSCULUS DOMAIN-ASSOCIATED PROTEIN 100683 XM_917317 MUS TRANSFORMATION/TRANSCRIPTION MUSCULUS DOMAIN-ASSOCIATED PROTEIN 100683 XM_899733 MUS TRANSFORMATION/TRANSCRIPTION MUSCULUS DOMAIN-ASSOCIATED PROTEIN 100683 XM_925612 MUS TRANSFORMATION/TRANSCRIPTION MUSCULUS DOMAIN-ASSOCIATED PROTEIN 108989 NM_133780 MUS TRANSLOCATED PROMOTER REGION MUSCULUS 17112 NM_008536 MUS TRANSMEMBRANE 4 SUPERFAMILY MUSCULUS MEMBER 1 21848 NM_145076 MUS SP. TRIPARTITE MOTIF PROTEIN 24 21848 NM_145076 MUS TRIPARTITE MOTIF PROTEIN 24 MUSCULUS 19720 NM_009054 MUS TRIPARTITE MOTIF PROTEIN 27 MUSCULUS 13867 NM_010153 MUS V-ERB-B2 ERYTHROBLASTIC LEUKEMIA MUSCULUS VIRAL ONCOGENE HOMOLOG 3 (AVIAN) 16653 NM_010937 MUS V-KI-RAS2 KIRSTEN RAT SARCOMA VIRAL MUSCULUS ONCOGENE HOMOLOG 16653 NM_021284 MUS V-KI-RAS2 KIRSTEN RAT SARCOMA VIRAL MUSCULUS ONCOGENE HOMOLOG 69922 NM_027260 MUS VACCINIA RELATED KINASE 2 MUSCULUS 233405 NM_178070 MUS VACUOLAR PROTEIN SORTING 33B MUSCULUS (YEAST) 22320 NM_016794 MUS VESICLE-ASSOCIATED MEMBRANE MUSCULUS PROTEIN 8 30960 NM_013933 MUS VESICLE-ASSOCIATED MEMBRANE MUSCULUS PROTEIN, ASSOCIATED PROTEIN A 218456 N/A MUS SIMILAR TO NUCLEOSIDE DIPHOSPHATE MUSCULUS KINASE B (NDK B) (NDP KINASE B) (P18 381082 N/A MUS SIMILAR TO MITOGEN-ACTIVATED MUSCULUS PROTEIN KINASE 14 ISOFORM 1; CYTOKINE SUP 381309 N/A MUS SIMILAR TO CDC42-BINDING PROTEIN MUSCULUS KINASE ALPHA; MYTONIC DYSTROPHY KINAS 384481 N/A MUS SIMILAR TO URIDINE MONOPHOSPHATE MUSCULUS KINASE

Example 2

Table 2 shows the results of a screen that led to accumulation of p62. The results are presented in a positive to negative ranking according to p62.

TABLE 2 Symbol Gene ID Gene rank Chmp4b 75608 1 Kpnb1 16211 2 Irak1 16179 3 Rhov 228543 4 Gm5285 383956 5 Eef1a2 13628 6 Ulk1 22241 7 Epha5 13839 8 LUCIFERASE −14 9 Rhot1 59040 10 Stk35 67333 11 Gtpbp4 69237 12 Prkaa2 108079 13 Pfkfb4 270198 14 Map3k14 53859 15 Rhog 56212 16 Mylk4 238564 17 Rhoc 11853 18 Cdc42bpg 240505 19 GeneID: 245619 245619 20 Tssk3 58864 21 Ap1s1 11769 22 Smok2a 27263 23 Agap3 213990 24 Shpk 74637 25 Cdk3 69681 26 N4bp2 333789 27 Ap3m2 64933 28 Rab20 19332 29 Vps4b 20479 30 Hgs 15239 31 Pik3c2a 18704 32 Etnk1 75320 33 Sh3gl2 20404 34 Prkar2b 19088 35 Rac1 19353 36 Gpn1 74254 37 Nkiras2 71966 38 Pik3cg 30955 39 Prkag1 19082 40 Phkg1 18682 41 GFP −10 42 Vps13a 271564 43 Txk 22165 44 Dab2 13132 45 Mast3 234385 46 Pdgfrl 68797 47 Vps36 70160 48 GeneID: 233024 #N/A 49 Prkaa1 105787 50 Pdpk1 18607 51 Doc2b 13447 52 Gm5374 385049 53 Nuak1 77976 54 Acvr2a 11480 55 Snx3 54198 56 Mapk9 26420 57 Camk4 12326 58 Grk6 26385 59 Nme2 18103 60 Pak4 70584 61 Stradb 227154 62 Rab19 19331 63 Pi4ka 224020 64 Camk2g 12325 65 Dlg4 13385 66 Cdadc1 71891 67 Mapkapk3 102626 68 Tjp2 21873 69 Gm318 240091 70 Cdkl5 382253 71 Snx16 74718 72 Cdc2l1 12537 73 Trpm6 225997 74 Ap1g1 11765 75 Rab14 68365 76 RFP −12 77 Pmvk 68603 78 Pank3 211347 79 Pkmyt1 268930 80 Crkl 12929 81 Fastkd5 380601 82 Etnk2 214253 83 Ephb6 13848 84 GeneID: 229309 #N/A 85 Aldh18a1 56454 86 Ak1 11636 87 Eif2ak1 15467 88 Kdr 16542 89 Gsk3a 606496 90 Gck 103988 91 Araf 11836 92 Arf5 11844 93 Dgkb 217480 94 Ltk 17005 95 Rab8a 17274 96 Ryk 20187 97 Gm4862 229879 98 Mknk2 17347 99 GeneID: 243968 #N/A 100 Arpc1a 56443 101 2310079N02Rik 66566 102 GeneID: 381981 #N/A 103 Nme7 171567 104 Ak2 11637 105 Tesk2 230661 106 Sik1 17691 107 Synj2 20975 108 Chek1 12649 109 GeneID: 384257 #N/A 110 Mapk3 26417 111 Itpka 228550 112 Gm1078 381835 113 Ciita 12265 114 GeneID: 381061 #N/A 115 Uck1 22245 116 Rps6ka2 20112 117 Prps111 75456 118 Rap1a 109905 119 Rac3 170758 120 Gm4776 212225 121 Eif2s3y 26908 122 Trpd5213 66745 123 GeneID: 245068 #N/A 124 lacZ −15 125 Sec23a 20334 126 Arfrp1 76688 127 Ras110b 276952 128 Hspb8 80888 129 Rab3d 19340 130 Arl4d 80981 131 GeneID: 384894 #N/A 132 GeneID: 381446 #N/A 133 GeneID: 268321 #N/A 134 Anxa7 11750 135 Hras1 15461 136 Wnk4 69847 137 Melk 17279 138 Frk 14302 139 Mapkapk2 17164 140 Rragc 54170 141 Phka1 18679 142 Becn1 56208 143 Pik3c2g 18705 144 Acvr2b 11481 145 Tie1 21846 146 Camk1g 215303 147 Phkg2 68961 148 Csnk1g1 214897 149 Khk 16548 150 Fgfr4 14186 151 Gucy2e 14919 152 Gm1893 381599 153 Rras2 66922 154 Ikbkb 16150 155 Gsg2 14841 156 Tspan7 21912 157 Keap1 50868 158 Rab6b 270192 159 Ern1 78943 160 Golga5 27277 161 Mlkl 74568 162 Epha6 13840 163 Ephb2 13844 164 Camkk1 55984 165 Psmc1 19179 166 Ankk1 244859 167 B2m 12010 168 Nkiras1 69721 169 Rab15 104886 170 Smg1 233789 171 Ckmt2 76722 172 Gm9824 432447 173 Nek4 23955 174 Trp53rk 76367 175 Mtor 56717 176 Vps39 269338 177 Rps6kl1 238323 178 Drg2 13495 179 Mark4 232944 180 Syt15 319508 181 Rerg 232441 182 Hipk2 15258 183 Cav3 12391 184 Grk5 14773 185 Map2k3 26397 186 Smok4a 272667 187 Rab7 19349 188 2810408M09Rik 381406 189 Chmp5 76959 190 Prkcd 18753 191 Snx2 67804 192 Mst1r 19882 193 Stk19 54402 194 Gk2 14626 195 Acvrl1 11482 196 Syt8 55925 197 Cyth3 19159 198 Tspan6 56496 199 Irak3 73914 200

TABLE 4 GeneID RefSeq Species Description 18607 NM_011062 MUS 3-PHOSPHOINOSITIDE DEPENDENT MUSCULUS PROTEIN KINASE-1 270198 NM_173019 MUS 6-PHOSPHOFRUCTO-2- MUSCULUS KINASE/FRUCTOSE-2,6- BIPHOSPHATASE 4 270198 NM_001039217 MUS 6-PHOSPHOFRUCTO-2- MUSCULUS KINASE/FRUCTOSE-2,6- BIPHOSPHATASE 4 270198 NM_001039216 MUS 6-PHOSPHOFRUCTO-2- MUSCULUS KINASE/FRUCTOSE-2,6- BIPHOSPHATASE 4 270198 NM_001039215 MUS 6-PHOSPHOFRUCTO-2- MUSCULUS KINASE/FRUCTOSE-2,6- BIPHOSPHATASE 4 56443 NM_019767 MUS ACTIN RELATED PROTEIN ⅔ MUSCULUS COMPLEX, SUBUNIT 1A 11482 NM_009612 MUS ACTIVIN A RECEPTOR, TYPE II-LIKE 1 MUSCULUS 11480 NM_007396 MUS ACTIVIN RECEPTOR IIA MUSCULUS 11481 NM_007397 MUS ACTIVIN RECEPTOR IIB MUSCULUS 11765 NM_009677 MUS ADAPTOR PROTEIN COMPLEX AP-1, MUSCULUS GAMMA 1 SUBUNIT 11769 NM_007457 MUS ADAPTOR PROTEIN COMPLEX AP-1, MUSCULUS SIGMA 1 64933 NM_029505 MUS ADAPTOR-RELATED PROTEIN MUSCULUS COMPLEX 3, MU 2 SUBUNIT 11636 NM_021515 MUS ADENYLATE KINASE 1 MUSCULUS 11637 NM_001033966 MUS ADENYLATE KINASE 2 MUSCULUS 11637 NM_016895 MUS ADENYLATE KINASE 2 MUSCULUS 11844 NM_007480 MUS ADP-RIBOSYLATION FACTOR 5 MUSCULUS 76688 NM_029702 MUS ADP-RIBOSYLATION FACTOR MUSCULUS RELATED PROTEIN 1 80981 NM_031160 MUS ADP-RIBOSYLATION FACTOR-LIKE 4D MUSCULUS 56454 NM_019698 MUS ALDEHYDE DEHYDROGENASE 18 MUSCULUS FAMILY, MEMBER A1 56454 NM_153554 MUS ALDEHYDE DEHYDROGENASE 18 MUSCULUS FAMILY, MEMBER A1 227154 NM_172656 MUS AMYOTROPHIC LATERAL SCLEROSIS 2 MUSCULUS (JUVENILE) CHROMOSOME REGION, CANDIDAT . . . 244859 NM_172922 MUS ANKYRIN REPEAT AND KINASE MUSCULUS DOMAIN CONTAINING 1 11750 NM_009674 MUS ANNEXIN A7 MUSCULUS 333789 NM_001024917 MUS BCL3 BINDING PROTEIN MUSCULUS 56208 NM_019584 MUS BECLIN 1 (COILED-COIL, MYOSIN-LIKE MUSCULUS BCL2-INTERACTING PROTEIN) 12010 NM_009735 MUS BETA-2 MICROGLOBULIN SPRETUS 12010 NM_009735 MUS BETA-2 MICROGLOBULIN MUSCULUS 215303 NM_144817 MUS CALCIUM/CALMODULIN-DEPENDENT MUSCULUS PROTEIN KINASE I GAMMA 12325 NM_178597 MUS CALCIUM/CALMODULIN-DEPENDENT MUSCULUS PROTEIN KINASE II GAMMA 12325 NM_001039139 MUS CALCIUM/CALMODULIN-DEPENDENT MUSCULUS PROTEIN KINASE II GAMMA 12325 NM_001039138 MUS CALCIUM/CALMODULIN-DEPENDENT MUSCULUS PROTEIN KINASE II GAMMA 12326 NM_009793 MUS CALCIUM/CALMODULIN-DEPENDENT MUSCULUS PROTEIN KINASE IV 55984 NM_018883 MUS CALCIUM/CALMODULIN-DEPENDENT MUSCULUS PROTEIN KINASE KINASE 1, ALPHA 74637 NM_029031 MUS CARBOHYDRATE KINASE-LIKE MUSCULUS 214897 NM_173185 MUS CASEIN KINASE 1, GAMMA 1 MUSCULUS 12391 NM_007617 MUS CAVEOLIN 3 MUSCULUS 240505 NM_001033342 MUS CDC42 BINDING PROTEIN KINASE XM_906449 MUSCULUS GAMMA (DMPK-LIKE) 240505 NM_001033342 MUS CDC42 BINDING PROTEIN KINASE XM_140553 MUSCULUS GAMMA (DMPK-LIKE) 12537 NM_007661 MUS CELL DIVISION CYCLE 2 HOMOLOG (S. POMBE)- MUSCULUS LIKE 1 213990 NM_139153 MUS CENTAURIN, GAMMA 3 MUSCULUS 12649 NM_007691 MUS CHECKPOINT KINASE 1 HOMOLOG (S. POMBE) MUSCULUS 13848 NM_007680 MUS CHICKEN EPH/ELK RECEPTOR-LIKE MUSCULUS PROTEIN 75608 NM_029362 MUS CHROMATIN MODIFYING PROTEIN 4B MUSCULUS 76959 NM_029814 MUS CHROMATIN MODIFYING PROTEIN 5 MUSCULUS 12265 NM_007575 MUS CLASS II TRANSACTIVATOR MUSCULUS 76722 NM_198415 MUS CREATINE KINASE, MITOCHONDRIAL 2 MUSCULUS 69681 NM_027165 MUS CYCLIN-DEPENDENT KINASE 3 MUSCULUS 382253 XM_912167 MUS CYCLIN-DEPENDENT KINASE-LIKE 5 NM_001024624 MUSCULUS NM_027986 XM_914101 XM_001000245 XM_001000259 XM_001000276 XM_001000293 71891 XM_001000308 MUS CYTIDINE AND DCMP DEAMINASE XM_001000321 MUSCULUS DOMAIN CONTAINING 1 XM_001000336 XM_001002774 XM_127813 XM_914101 XM_985192 71891 XM_901860 MUS CYTIDINE AND DCMP DEAMINASE MUSCULUS DOMAIN CONTAINING 1 71891 XM_923095 MUS CYTIDINE AND DCMP DEAMINASE MUSCULUS DOMAIN CONTAINING 1 71891 XM_894723 MUS CYTIDINE AND DCMP DEAMINASE MUSCULUS DOMAIN CONTAINING 1 71891 XM_923089 MUS CYTIDINE AND DCMP DEAMINASE MUSCULUS DOMAIN CONTAINING 1 71891 XM_901847 MUS CYTIDINE AND DCMP DEAMINASE MUSCULUS DOMAIN CONTAINING 1 71891 XM_901853 MUS CYTIDINE AND DCMP DEAMINASE MUSCULUS DOMAIN CONTAINING 1 71891 XM_923082 MUS CYTIDINE AND DCMP DEAMINASE MUSCULUS DOMAIN CONTAINING 1 71891 XM_923072 MUS CYTIDINE AND DCMP DEAMINASE MUSCULUS DOMAIN CONTAINING 1 71891 XM_923086 MUS CYTIDINE AND DCMP DEAMINASE MUSCULUS DOMAIN CONTAINING 1 71891 XM_901857 MUS CYTIDINE AND DCMP DEAMINASE MUSCULUS DOMAIN CONTAINING 1 71891 XM_127813 MUS CYTIDINE AND DCMP DEAMINASE MUSCULUS DOMAIN CONTAINING 1 71891 XM_901866 MUS CYTIDINE AND DCMP DEAMINASE MUSCULUS DOMAIN CONTAINING 1 71891 XM_923093 MUS CYTIDINE AND DCMP DEAMINASE MUSCULUS DOMAIN CONTAINING 1 13495 NM_021354 MUS DEVELOPMENTALLY REGULATED GTP MUSCULUS BINDING PROTEIN 2 217480 NM_178681 MUS DIACYLGLYCEROL KINASE, BETA MUSCULUS 13132 NM_001037905 MUS DISABLED HOMOLOG 2 (DROSOPHILA) MUSCULUS 13132 NM_001008702 MUS DISABLED HOMOLOG 2 (DROSOPHILA) MUSCULUS 13132 NM_023118 MUS DISABLED HOMOLOG 2 (DROSOPHILA) MUSCULUS 13385 NM_007864 MUS DISCS, LARGE HOMOLOG 4 MUSCULUS (DROSOPHILA) 13447 NM_007873 MUS DOUBLE C2, BETA MUSCULUS 13839 NM_007937 MUS ECK-LIKE SEQUENCE 1 MUSCULUS 78943 NM_023913 MUS ENDOPLASMIC RETICULUM (ER) TO MUSCULUS NUCLEUS SIGNALLING 1 13840 NM_007938 MUS EPH RECEPTOR A6 MUSCULUS 13844 NM_010142 MUS EPH RECEPTOR B2 MUSCULUS 75320 XM_284250 MUS ETHANOLAMINE KINASE 1 MUSCULUS 75320 NM_029250 MUS ETHANOLAMINE KINASE 1 XM_908334 MUSCULUS XM_979562 214253 NM_175443 MUS ETHANOLAMINE KINASE 2 MUSCULUS 13628 NM_007906 MUS EUKARYOTIC TRANSLATION MUSCULUS ELONGATION FACTOR 1 ALPHA 2 15467 NM_013557 MUS EUKARYOTIC TRANSLATION MUSCULUS INITIATION FACTOR 2 ALPHA KINASE 1 26908 NM_012011 MUS EUKARYOTIC TRANSLATION MUSCULUS INITIATION FACTOR 2, SUBUNIT 3, STRUCTURAL GENE . . . 18103 NM_008705 MUS EXPRESSED IN NON-METASTATIC MUSCULUS CELLS 2, PROTEIN 380601 NM_198176 MUS EXPRESSED SEQUENCE C78212 MUSCULUS 14186 NM_008011 MUS FIBROBLAST GROWTH FACTOR MUSCULUS RECEPTOR 4 56717 NM_020009 MUS FK506 BINDING PROTEIN 12- MUSCULUS RAPAMYCIN ASSOCIATED PROTEIN 1 56717 NM_001039554 MUS FK506 BINDING PROTEIN 12- MUSCULUS RAPAMYCIN ASSOCIATED PROTEIN 1 14302 NM_010237 MUS FYN-RELATED KINASE MUSCULUS 14773 NM_018869 MUS G PROTEIN-COUPLED RECEPTOR MUSCULUS KINASE 5 26385 NM_011938 MUS G PROTEIN-COUPLED RECEPTOR MUSCULUS KINASE 6 26385 NM_001038018 MUS G PROTEIN-COUPLED RECEPTOR MUSCULUS KINASE 6 381835 XM_355840 MUS GENE MODEL 1078, (NCBI) MUSCULUS 381835 XM_911944 MUS GENE MODEL 1078, (NCBI) MUSCULUS 381599 XM_355556 MUS GENE MODEL 1893, (NCBI) MUSCULUS 240091 XM_139919 MUS GENE MODEL 318, (NCBI) [replaced XM_619462 MUSCULUS with XM_001481303 545204] XM_622850 14841 NM_010353 MUS GERM CELL-SPECIFIC GENE 2 MUSCULUS 103988 NM_010292 MUS GLUCOKINASE MUSCULUS 14626 NM_010294 MUS GLYCEROL KINASE 2 MUSCULUS 606496 NM_001031667 MUS GLYCOGEN SYNTHASE KINASE 3 MUSCULUS ALPHA 27277 NM_013747 MUS GOLGI AUTOANTIGEN, GOLGIN MUSCULUS SUBFAMILY A, 5 69237 NM_027000 MUS GTP BINDING PROTEIN 4 MUSCULUS 14919 NM_008192 MUS GUANYLATE CYCLASE 2E MUSCULUS 15461 NM_008284 MUS SP. HARVEY RAT SARCOMA VIRUS ONCOGENE 1 15461 NM_008284 MUS HARVEY RAT SARCOMA VIRUS MUSCULUS ONCOGENE 1 80888 NM_030704 MUS HEAT SHOCK PROTEIN 8 MUSCULUS 15239 NM_008244 MUS HGF-REGULATED TYROSINE KINASE MUSCULUS SUBSTRATE 15258 NM_010433 MUS HOMEODOMAIN INTERACTING MUSCULUS PROTEIN KINASE 2 212225 NM_172504 MUS HYPOTHETICAL PROTEIN 4930509O22 MUSCULUS 16150 NM_010546 MUS INHIBITOR OF KAPPAB KINASE BETA MUSCULUS 228550 NM_146125 MUS INOSITOL 1,4,5-TRISPHOSPHATE 3- MUSCULUS KINASE A 16179 NM_008363 MUS INTERLEUKIN-1 RECEPTOR- MUSCULUS ASSOCIATED KINASE 1 73914 NM_028679 MUS INTERLEUKIN-1 RECEPTOR- MUSCULUS ASSOCIATED KINASE 3 16211 NM_008379 MUS KARYOPHERIN (IMPORTIN) BETA 1 MUSCULUS 50868 NM_016679 MUS KELCH-LIKE ECH-ASSOCIATED MUSCULUS PROTEIN 1 16548 NM_008439 MUS KETOHEXOKINASE MUSCULUS 16542 NM_010612 MUS KINASE INSERT DOMAIN PROTEIN MUSCULUS RECEPTOR 16542 NM_010612 MUS SP. KINASE INSERT DOMAIN PROTEIN RECEPTOR 17005 NM_008523 MUS LEUKOCYTE TYROSINE KINASE MUSCULUS 17005 NM_206942 MUS LEUKOCYTE TYROSINE KINASE MUSCULUS 17005 NM_206941 MUS LEUKOCYTE TYROSINE KINASE MUSCULUS 17005 NM_203345 MUS LEUKOCYTE TYROSINE KINASE MUSCULUS 19882 NM_009074 MUS SP. MACROPHAGE STIMULATING 1 RECEPTOR (C-MET-RELATED TYROSINE KINASE) 19882 NM_009074 MUS MACROPHAGE STIMULATING 1 MUSCULUS RECEPTOR (C-MET-RELATED TYROSINE KINASE) 17164 NM_008551 MUS MAP KINASE-ACTIVATED PROTEIN MUSCULUS KINASE 2 17347 NM_021462 MUS MAP KINASE-INTERACTING MUSCULUS SERINE/THREONINE KINASE 2 232944 NM_172279 MUS MAP/MICROTUBULE AFFINITY- MUSCULUS REGULATING KINASE 4 17279 NM_010790 MUS MATERNAL EMBRYONIC LEUCINE MUSCULUS ZIPPER KINASE 268930 NM_023058 MUS MEMBRANE-ASSOCIATED TYROSINE- MUSCULUS AND THREONINE-SPECIFIC CDC2- INHIBITORY KI . . . 234385 NM_199308 MUS MICROTUBULE ASSOCIATED [replaced XM_134245 MUSCULUS SERINE/THREONINE KINASE 3 with XM_913385 546071] 234385 XM_888290 MUS MICROTUBULE ASSOCIATED MUSCULUS SERINE/THREONINE KINASE 3 234385 XM_922751 MUS MICROTUBULE ASSOCIATED MUSCULUS SERINE/THREONINE KINASE 3 234385 XM_897983 MUS MICROTUBULE ASSOCIATED MUSCULUS SERINE/THREONINE KINASE 3 234385 XM_897989 MUS MICROTUBULE ASSOCIATED MUSCULUS SERINE/THREONINE KINASE 3 234385 XM_620670 MUS MICROTUBULE ASSOCIATED MUSCULUS SERINE/THREONINE KINASE 3 234385 XM_897962 MUS MICROTUBULE ASSOCIATED MUSCULUS SERINE/THREONINE KINASE 3 234385 XM_897954 MUS MICROTUBULE ASSOCIATED MUSCULUS SERINE/THREONINE KINASE 3 26420 NM_016961 MUS MITOGEN ACTIVATED PROTEIN MUSCULUS KINASE 9 26420 NM_207692 MUS MITOGEN ACTIVATED PROTEIN MUSCULUS KINASE 9 26397 NM_008928 MUS MITOGEN ACTIVATED PROTEIN MUSCULUS KINASE KINASE 3 53859 NM_016896 MUS MITOGEN-ACTIVATED PROTEIN MUSCULUS KINASE KINASE KINASE 14 102626 NM_178907 MUS MITOGEN-ACTIVATED PROTEIN MUSCULUS KINASE-ACTIVATED PROTEIN KINASE 3 74568 NM_029005 MUS MIXED LINEAGE KINASE DOMAIN- XM_001003995 MUSCULUS LIKE XM_001003998 XM_356104 XM_895027 XM_916960 XM_924589 XM_924589 74568 XM_895027 MUS MIXED LINEAGE KINASE DOMAIN- MUSCULUS LIKE 74568 XM_902435 MUS MIXED LINEAGE KINASE DOMAIN- MUSCULUS LIKE 74568 XM_916960 MUS MIXED LINEAGE KINASE DOMAIN- MUSCULUS LIKE 74568 XM_356104 MUS MIXED LINEAGE KINASE DOMAIN- MUSCULUS LIKE 74568 XM_924585 MUS MIXED LINEAGE KINASE DOMAIN- MUSCULUS LIKE 69721 NM_023526 MUS NFKB INHIBITOR INTERACTING RAS- MUSCULUS LIKE PROTEIN 1 71966 NM_028024 MUS NFKB INHIBITOR INTERACTING RAS- MUSCULUS LIKE PROTEIN 2 23955 NM_011849 MUS NIMA (NEVER IN MITOSIS GENE A)- MUSCULUS RELATED EXPRESSED KINASE 4 171567 NM_178071 MUS NON-METASTATIC CELLS 7, PROTEIN MUSCULUS EXPRESSED IN 171567 NM_138314 MUS NON-METASTATIC CELLS 7, PROTEIN MUSCULUS EXPRESSED IN 70584 NM_027470 MUS P21 (CDKN1A)-ACTIVATED KINASE 4 MUSCULUS 211347 NM_145962 MUS PANTOTHENATE KINASE 3 MUSCULUS 18704 NM_011083 MUS PHOSPHATIDYLINOSITOL 3-KINASE, MUSCULUS C2 DOMAIN CONTAINING, ALPHA POLYPEPTIDE 18705 NM_207683 MUS PHOSPHATIDYLINOSITOL 3-KINASE, MUSCULUS C2 DOMAIN CONTAINING, GAMMA POLYPEPTIDE 18705 NM_011084 MUS PHOSPHATIDYLINOSITOL 3-KINASE, MUSCULUS C2 DOMAIN CONTAINING, GAMMA POLYPEPTIDE 224020 NM_001001983 MUS PHOSPHATIDYLINOSITOL 4-KINASE, MUSCULUS CATALYTIC, ALPHA POLYPEPTIDE 30955 NM_020272 MUS PHOSPHOINOSITIDE-3-KINASE, MUSCULUS CATALYTIC, GAMMA POLYPEPTIDE 68603 NM_026784 MUS PHOSPHOMEVALONATE KINASE MUSCULUS 18679 NM_008832 MUS PHOSPHORYLASE KINASE ALPHA 1 MUSCULUS 18679 NM_173021 MUS PHOSPHORYLASE KINASE ALPHA 1 MUSCULUS 18682 NM_011079 MUS PHOSPHORYLASE KINASE GAMMA 1 MUSCULUS 68961 NM_026888 MUS PHOSPHORYLASE KINASE, GAMMA 2 MUSCULUS (TESTIS) 68797 NM_026840 MUS PLATELET-DERIVED GROWTH FACTOR MUSCULUS RECEPTOR-LIKE 19159 NM_011182 MUS PLECKSTRIN HOMOLOGY, SEC7 AND MUSCULUS COILED-COIL DOMAINS 3 19179 NM_008947 MUS PROTEASE (PROSOME, MACROPAIN) MUSCULUS 26S SUBUNIT, ATPASE 1 18753 NM_011103 MUS PROTEIN KINASE C, DELTA MUSCULUS 105787 NM_001013367 MUS PROTEIN KINASE, AMP-ACTIVATED, MUSCULUS ALPHA 1 CATALYTIC SUBUNIT 108079 NM_178143 MUS PROTEIN KINASE, AMP-ACTIVATED, MUSCULUS ALPHA 2 CATALYTIC SUBUNIT 19082 NM_016781 MUS PROTEIN KINASE, AMP-ACTIVATED, MUSCULUS GAMMA 1 NON-CATALYTIC SUBUNIT 19088 NM_011158 MUS PROTEIN KINASE, CAMP DEPENDENT MUSCULUS REGULATORY, TYPE II BETA 26417 NM_011952 MUS SP. PROTEIN KINASE, MITOGEN ACTIVATED KINASE 3 26417 NM_011952 MUS PROTEIN KINASE, MITOGEN MUSCULUS ACTIVATED KINASE 3 68365 NM_026697 MUS RAB14, MEMBER RAS ONCOGENE MUSCULUS FAMILY 104886 NM_134050 MUS RAB15, MEMBER RAS ONCOGENE MUSCULUS FAMILY 19331 NM_011226 MUS RAB19, MEMBER RAS ONCOGENE MUSCULUS FAMILY 19332 NM_011227 MUS RAB20, MEMBER RAS ONCOGENE MUSCULUS FAMILY 19340 NM_031874 MUS RAB3D, MEMBER RAS ONCOGENE MUSCULUS FAMILY 270192 NM_173781 MUS RAB6B, MEMBER RAS ONCOGENE MUSCULUS FAMILY 19349 NM_009005 MUS RAB7, MEMBER RAS ONCOGENE MUSCULUS FAMILY 17274 NM_023126 MUS SP. RAB8A, MEMBER RAS ONCOGENE FAMILY 17274 NM_023126 MUS RAB8A, MEMBER RAS ONCOGENE MUSCULUS FAMILY 11853 NM_007484 MUS RAS HOMOLOG GENE FAMILY, MUSCULUS MEMBER C 56212 NM_019566 MUS RAS HOMOLOG GENE FAMILY, MUSCULUS MEMBER G 59040 NM_021536 MUS RAS HOMOLOG GENE FAMILY, MUSCULUS MEMBER T1 228543 NM_145530 MUS RAS HOMOLOG GENE FAMILY, MUSCULUS MEMBER V 232441 NM_181988 MUS RAS-LIKE, ESTROGEN-REGULATED, MUSCULUS GROWTH-INHIBITOR 276952 NM_001013386 MUS RAS-LIKE, FAMILY 10, MEMBER B MUSCULUS 19353 NM_009007 MUS RAS-RELATED C3 BOTULINUM MUSCULUS SUBSTRATE 1 170758 NM_133223 MUS RAS-RELATED C3 BOTULINUM MUSCULUS SUBSTRATE 3 54170 NM_017475 MUS RAS-RELATED GTP BINDING C MUSCULUS 109905 NM_145541 MUS RAS-RELATED PROTEIN-1A MUSCULUS 20187 NM_013649 MUS SP. RECEPTOR-LIKE TYROSINE KINASE 20187 NM_013649 MUS RECEPTOR-LIKE TYROSINE KINASE MUSCULUS 66922 NM_025846 MUS RELATED RAS VIRAL (R-RAS) MUSCULUS ONCOGENE HOMOLOG 2 20112 NM_011299 MUS RIBOSOMAL PROTEIN S6 KINASE, MUSCULUS RELATED SEQUENCE 1 238323 NM_146244 MUS RIBOSOMAL PROTEIN S6 KINASE-LIKE 1 MUSCULUS 75456 NM_029294 MUS RIKEN CDNA 1700011K15 GENE MUSCULUS 66566 NM_025636 MUS RIKEN CDNA 2310079N02 GENE MUSCULUS 233789 NM_001031814 MUS RIKEN CDNA 2610207I05 GENE MUSCULUS 272667 XM_895217 MUS RIKEN CDNA 4930513D10 GENE MUSCULUS 272667 XM_142762 MUS RIKEN CDNA 4930513D10 GENE MUSCULUS 272667 XM_912174 MUS RIKEN CDNA 4930513D10 GENE MUSCULUS 271564 NM_173028 MUS RIKEN CDNA 4930516E05 GENE MUSCULUS 20334 NM_009147 MUS SEC23A (S. CEREVISIAE) MUSCULUS 54402 NM_019442 MUS SERINE/THREONINE KINASE 19 MUSCULUS 67333 NM_001038635 MUS SERINE/THREONINE KINASE 35 MUSCULUS 67333 NM_183262 MUS SERINE/THREONINE KINASE 35 MUSCULUS 20404 NM_019535 MUS SH3-DOMAIN GRB2-LIKE 2 MUSCULUS 383956 XM_916921 MUS SIMILAR TO MAP/MICROTUBULE MUSCULUS AFFINITY-REGULATING KINASE 3 383956 XM_357348 MUS SIMILAR TO MAP/MICROTUBULE MUSCULUS AFFINITY-REGULATING KINASE 3 245068 XM_918167 MUS SIMILAR TO MAP/MICROTUBULE MUSCULUS AFFINITY-REGULATING KINASE 4 (MAP/MICROTUBU . . . 245068 XM_142402 MUS SIMILAR TO MAP/MICROTUBULE MUSCULUS AFFINITY-REGULATING KINASE 4 (MAP/MICROTUBU . . . 238564 NM_001166030 MUS SIMILAR TO MYOSIN LIGHT CHAIN XM_910560 MUSCULUS KINASE 238564 XM_111421 MUS SIMILAR TO MYOSIN LIGHT CHAIN MUSCULUS KINASE 229879 XM_143595 MUS GM4862 PREDICTED GENE 4862 XM_001478899 MUSCULUS SIMILAR TO NON-METASTATIC CELLS 2, PROTEIN (NM23B) EXPRESSED IN ISOFOR . . . GM5374 PREDICTED GENE 5374 385049 XM_904204 MUS SIMILAR TO RHO-ASSOCIATED XM_001473528 MUSCULUS COILED-COIL FORMING KINASE 1 385049 XM_358017 MUS SIMILAR TO RHO-ASSOCIATED MUSCULUS COILED-COIL FORMING KINASE 1 17691 NM_010831 MUS SNF1-LIKE KINASE MUSCULUS 74718 NM_029068 MUS SORTING NEXIN 16 MUSCULUS 67804 NM_026386 MUS SORTING NEXIN 2 MUSCULUS 54198 NM_017472 MUS SORTING NEXIN 3 MUSCULUS 27263 XM_889037 MUS SPERM MOTILITY KINASE 2 MUSCULUS 27263 XM_620264 MUS SPERM MOTILITY KINASE 2 MUSCULUS 27263 XM_135656 MUS SPERM MOTILITY KINASE 2 MUSCULUS 27263 XM_889060 MUS SPERM MOTILITY KINASE 2 MUSCULUS 27263 XM_620265 MUS SPERM MOTILITY KINASE 2 MUSCULUS 27263 XM_907097 MUS SPERM MOTILITY KINASE 2 MUSCULUS 20975 NM_011523 MUS SYNAPTOJANIN 2 MUSCULUS 55925 NM_018802 MUS SYNAPTOTAGMIN VIII MUSCULUS 319508 NM_176931 MUS SYNAPTOTAGMIN XV MUSCULUS 319508 NM_181529 MUS SYNAPTOTAGMIN XV MUSCULUS 230661 NM_146151 MUS TESTIS-SPECIFIC KINASE 2 MUSCULUS 58864 NM_080442 MUS TESTIS-SPECIFIC SERINE KINASE 3 MUSCULUS 56496 NM_019656 MUS TETRASPANIN 6 MUSCULUS 21912 NM_019634 MUS TETRASPANIN 7 MUSCULUS 21873 NM_011597 MUS TIGHT JUNCTION PROTEIN 2 MUSCULUS 225997 NM_153417 MUS TRANSIENT RECEPTOR POTENTIAL MUSCULUS CATION CHANNEL, SUBFAMILY M, MEMBER 6 381406 NM_023815 MUS TRP53 REGULATING KINASE MUSCULUS 76367 NM_023815 MUS TRP53 REGULATING KINASE MUSCULUS 76367 NM_001007581 MUS TRP53 REGULATING KINASE MUSCULUS 381406 NM_001007581 MUS TRP53 REGULATING KINASE MUSCULUS 66745 NM_025741 MUS TUMOR PROTEIN D52-LIKE 3 MUSCULUS 22165 NM_013698 MUS TXK TYROSINE KINASE MUSCULUS 21846 NM_011587 MUS TYROSINE KINASE RECEPTOR 1 MUSCULUS 21846 NM_011587 MUS SP. TYROSINE KINASE RECEPTOR 1 22241 NM_009469 MUS UNC-51 LIKE KINASE 1 (C. ELEGANS) MUSCULUS 22245 NM_011675 MUS URIDINE-CYTIDINE KINASE 1 MUSCULUS 12929 NM_007764 MUS V-CRK SARCOMA VIRUS CT10 MUSCULUS ONCOGENE HOMOLOG (AVIAN)-LIKE 11836 NM_009703 MUS V-RAF MURINE SARCOMA 3611 VIRAL MUSCULUS ONCOGENE HOMOLOG 70160 NM_027338 MUS VACUOLAR PROTEIN SORTING 36 MUSCULUS (YEAST) 20479 NM_009190 MUS VACUOLAR PROTEIN SORTING 4B MUSCULUS (YEAST) 269338 NM_178851 MUS VPS39 MUSCULUS 269338 NM_147153 MUS VPS39 MUSCULUS 69847 NM_175638 MUS WNK LYSINE DEFICIENT PROTEIN MUSCULUS KINASE 4 74254 NM_133756 MUS XPA BINDING PROTEIN 1 MUSCULUS 77976 NM_001004363 MUS ZNUAK FAMILY, SNF1-LIKE KINASE, 1 MUSCULUS 229309 N/A MUS SIMILAR TO PHOSPHOGLYCERATE MUSCULUS KINASE (EC 2.7.2.3) - MOUSE 243968 N/A MUS SIMILAR TO KIAA1883 PROTEIN MUSCULUS 245619 NM_027067 MUS GLYCOGEN SYNTHASE KINASE 3 [Replaced MUSCULUS ALPHA PROBABLE PSEUDOGENE with 69389] 268321 N/A MUS SIMILAR TO NUCLEOSIDE [Replaced MUSCULUS DIPHOSPHATE KINASE B with 432482] 381061 NM_013741 MUS SPERM MOTILITY KINASE 2A [Replaced MUSCULUS with 27263] 381446 N/A MUS SIMILAR TO PHOSPHOGLYCERATE MUSCULUS KINASE (EC 2.7.2.3) - MOUSE 381981 N/A MUS SIMILAR TO AARF DOMAIN MUSCULUS CONTAINING KINASE 4 384257 N/A MUS SIMILAR TO NUCLEOSIDE MUSCULUS DIPHOSPHATE KINASE B 384894 N/A MUS SIMILAR TO CYTOSOLIC THYMIDINE MUSCULUS KINASE 432447 N/A MUS PHOSPHATIDYLETHANOLAMINE- MUSCULUS BINDING PROTEIN PSEUDOGENE

In Tables 1 and 2, the numbers in the column labeled GeneID correspond with the accession numbers in the Entrez GeneID database made available by the National Center for Biotechnology Information (NCBI). The Entrez GeneID may be used to identify corresponding sequences such as, for example, genomic DNA, mRNA and protein sequences.

In Tables 3 and 4, each GeneID is presented along with its corresponding accession number(s) from the NCBI Reference Sequences (RefSeq) database for mRNA transcripts. Through the accession numbers, the sequences are readily available. Table 3 contains sequences from Table 1. Table 4 contains sequences from Table 2. The rank order of the GenelDs in Tables 1 and 2 are not maintained in Tables 3 and 4.

The terms and expressions which have been employed are used as terms of descriptions and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, it should be understood that although the present invention has been illustrated by specific embodiments and optional features, modification and/or variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

In addition, where features or aspects of the invention are described in terms of Markush group or other grouping of alternatives, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.

All references, including the disclosures of each patent, patent application, publication and accession number to database sequences, cited or described in this document are hereby incorporated herein by reference, in their entireties. 

What is claimed is:
 1. A method for identifying compounds that inhibit or stimulate the autophagy pathway for treatment of a disease state associated with an autophagy pathway defect, comprising measuring the effect of one or more test compounds on the inhibition or stimulation of a product of one or more of the genes or gene fragments identified in Table 3 or Table
 4. 2. A method for identifying individuals susceptible to or afflicted with a disease state associated with an autophagy pathway defect, comprising testing a biological sample from an individual for a characteristic of one or more polypeptides produced by expression of one or more of the genes or gene fragments identified in Table 3 or Table 4 that is indicative of said disease state, wherein said characteristic is selected from the presence of at least one of said polypeptides, the absence of at least one of said polypeptides, an elevated level of at least one of said polypeptides, a reduced level of at least one of said polypeptides and, for two or more of said polypeptides, combinations thereof.
 3. A device for detecting the expression of a plurality of autophagy-related genes associated with an autophagy pathway defect, said device comprising a substrate to which is affixed at known locations a plurality of probes, wherein the probes comprise: a) a plurality of oligonucleotides or polynucleotides, each of which specifically binds to a different sequence selected from any of the sequences identified in Table 3 or Table 4 or fragments thereof; or b) a plurality of polypeptide binding agents, each of which specifically binds to a different polypeptide or fragment thereof produced by expression of a gene or gene fragment comprising any of the sequences identified in Table 3 or Table 4 or fragments thereof.
 4. A kit for assaying the expression of autophagy-related genes associated with an autophagy pathway defect, comprising at least one container and a collection of two or more probes, wherein the probes comprise: a) oligonucleotides or polynucleotides that specifically bind to two or more genes or gene fragments comprising any of the sequences identified in Table 3 or Table 4, or fragments thereof; or b) polypeptide binding agents that specifically bind to polypeptides produced by expression of two or more genes or gene fragments comprising any of the sequences identified in Table 3 or Table 4, or fragments thereof. 